Patent application title: Sharply Resolving Labeled Protein Molecular Weight Standards
Inventors:
Roumen Bogoev (San Marcos, CA, US)
Douglas Kang (Rancho Santa Margarita, CA, US)
Assignees:
INVITROGEN CORPORATION
IPC8 Class: AG01N27447FI
USPC Class:
204461
Class name: Electrophoresis or electro-osmosis processes and electrolyte compositions therefor when not provided for elsewhere gel electrophoresis with analysis or detailed detection
Publication date: 2009-07-16
Patent application number: 20090178926
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Patent application title: Sharply Resolving Labeled Protein Molecular Weight Standards
Inventors:
Roumen BOGOEV
Douglas KANG
Agents:
INVITROGEN CORPORATION;C/O INTELLEVATE
Assignees:
INVITROGEN CORPORATION
Origin: MINNEAPOLIS, MN US
IPC8 Class: AG01N27447FI
USPC Class:
204461
Abstract:
Pre-labeled protein standards useful in electrophoresis that have sharp,
consistent separation characteristics that are substantially the same as
those of their unlabeled counterparts are provided. The invention
provides pre-labeled protein standard sets that include a plurality of
labeled proteins that are labeled on a first amino acid, in which side
reactions of the label with amino acids not targeted for labeling are
reduced.Claims:
1-89. (canceled)
90. A method of characterizing one or more proteins comprising: (a) electrophoresing one or more proteins and at least one prelabeled protein standard set of claim 1 or claim 25 in a gel; and (b) comparing the migration of the one or more proteins with the migration of least one protein standard of the pre-labeled standard set.
91. The method of claim 90, further comprising (c) determining the molecular weight of the one or more proteins.
92. The method of claim 90, wherein at least two of the two or more protein standards having molecular weights of 10 kDa or greater migrate within 5% of the distance of the that the same protein standard in unlabeled form migrates.
93. The method of claim 90, wherein the width of the bands on a gel of each of the electrophoresed protein standards of the set having a molecular weight of 10 kDa or greater does not vary by more than 2-fold.
94. The method of claim 93, wherein the band intensities of the protein standards of the pre-labeled protein standard set having molecular weights of 10 kDa or greater does not vary by more than 2.5 fold.
Description:
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001]This application claims benefit of priority to U.S. provisional application 60/820,101, filed Jul. 21, 2006 and to U.S. provisional application 60/870,252, filed Dec. 15, 2006, both of which are incorporated by reference in their entireties.
REFERENCE TO A SEQUENCE LISTING
[0002]This application incorporates by reference a Sequence Listing submitted with this application as text file IVGN 563_WorkFile.txt created on Jul. 21, 2007 and having a size of 87.3 kilobytes.
BACKGROUND OF THE INVENTION
[0003]1. Field of the Invention
[0004]The invention relates generally to labeled protein standards for use in biochemical separations and more specifically to labeled protein standards for used in gel electrophoresis.
[0005]2. Background Information
[0006]Tools that aid in the development of new drugs and new medical diagnostics, as well as certain diagnostics themselves, require accurate and efficient analysis of protein samples. This in turn requires markers that accurately allow the identification of the size of proteins in a protein sample that is separated using separation methods. Separation methods that are commonly performed in biochemistry for the purification, identification, and characterization of proteins include chromatography, gel electrophoresis, and solution electrophoresis. These methods typically use standards for molecular weight or charge determination. Gel electrophoresis in particular is a common tool for the development of new drugs and medical diagnostics that is typically performed with molecular weight markers.
[0007]Pre-labeled protein standards for electrophoresis are notoriously less sharply resolving than unlabeled standards, and often the molecular weights of the labeled markers are inexact, differing from the unlabeled proteins by varying amounts. The bands of a pre-stained protein marker run in a denaturing polyacrylamide gel can be, for example, significantly wider and more diffuse than a band that results from the same protein that has not been pre-labeled, but instead is stained after electrophoresis is complete. This is largely due to the difficulties in uniformly labeling a particular protein standard.
[0008]Labeling of proteins is typically performed by attaching a label to a chemical group of one or more amino acid residues of the protein. The significant reactive groups of amino acids behave as nucleophiles in chemical reactions, for example, the sulfhydryl group of cysteine; the amino group of an N-terminal amino acid or of lysine, histidine, tryptophan, or arginine; the carboxyl group of aspartate and glutamate or a C-terminal amino acid; the phenolate of tyrosine; and the thioether of methionine. The selection of a particular reactive chemical group on the dye to be conjugated to a protein and manipulation of reaction conditions at which a chemical conjugation is performed (such as, for example, pH) will typically favor conjugation of a dye to one or more particular amino acids.
[0009]Although reaction conditions can be adjusted to reduce side reactions with one or more amino acids that are not targeted for labeling, side reactions are difficult to completely eliminate or control. The addition of label to a variable number of sites of a particular protein through side reactions reduces the uniformity in the amount of label attached to the protein, such that a given labeled protein standard comprises a population of labeled protein molecules in which different members of the population have different migration characteristics. Pre-labeled standards therefore typically do not resolve as well as unlabeled proteins in separations, producing bands on electrophoresis gels, for example, that are much less sharp than the bands produced by the same proteins electrophoresed in unlabeled form. The variability of labeling of pre-labeled standards often makes molecular weight determination using pre-labeled standards unreliable.
[0010]Another factor contributing to poor resolution of pre-labeled proteins on electrophoresis gels is protein-to-protein variability in the ratio of the number of attached dye molecules to molecular weight. Because a protein standard set uses different marker proteins to represent different molecular weights, and the different proteins of the set have variable ratios of the number of target amino acid residues to molecular weight, it is often necessary to mix different amounts of individual labeled protein standards to provide a pre-labeled marker set having proteins with similar intensity for visualization of the marker proteins. In many cases, this requires that one or more labeled proteins will be "overloaded" in a gel lane with respect to protein amount to achieve a desirable intensity for the resulting band on an electrophoresis gel. The overloading of proteins of the standard set leads to bands on the gel that are broad and not sharply delineated, making it difficult to assess the migration distance of the protein of a particular molecular weight.
SUMMARY OF THE INVENTION
[0011]Provided herein are labeled protein standards useful in electrophoresis or chromatography that have consistent separation characteristics that are substantially the same as the separation characteristics of their unlabeled counterparts. The invention provides pre-labeled protein standard sets that include a plurality of labeled proteins, in which one or more of the labeled proteins is selectively labeled on a first amino acid. A protein that is selectively labeled with a labeling compound on a first, or target, amino acid has a labeling compound conjugated to the first amino acid and is either: depleted in residues of a second, or non-target, amino acid that can react with the labeling compound; or: includes an amino acid sequence having homology to a naturally-occurring protein, in which the sequence has fewer residues of a second amino acid that is capable of reacting with the labeling compound than the wild-type protein sequence from which it is derived.
[0012]By reducing the number of residues of amino acids that can bind a labeling compound in side reactions, variability in the amount of labeling compound attached to a given protein molecule is reduced. The reduction in multiple species of a labeled protein that would otherwise result from this labeling variability provides for more precise separation characteristics. The present invention provides pre-labeled protein standard sets that when electrophoresed give sharp bands that have migration distances consistent with the migration distances of the proteins of the standard set electrophoresed in unlabeled form.
[0013]In certain embodiments, a labeling compound conjugated to a first amino acid is a dye. The dye can comprise a chromophore or fluorophore. Reducing or eliminating the attachment of a dye to residues of one or more amino acids not targeted for labeling decreases variability in the amount and position of dye attached to a marker protein. The specificity of labeling achieved using the methods provided in the invention produces labeled proteins that are highly-resolving in separation procedures, such as electrophoresis on denaturing gels.
[0014]In one aspect, the invention provides a pre-labeled protein standard set comprising a plurality of labeled proteins, in which one or more of the proteins of the plurality is selectively labeled, in which a selectively labeled protein comprises a labeling compound on a first, or target, amino acid, and has less than one residue of a second amino acid that reacts with the labeling compound per ten kilodaltons (kDa) of protein. In some embodiments, a selectively labeled protein of the invention lacks residues of a second amino acid that can react with a labeling compound.
[0015]In some embodiments of this aspect of the invention, a selectively labeled protein includes an amino acid sequence having homology to an amino acid sequence of a naturally-occurring protein, in which the naturally-occurring protein is naturally depleted in or deficient in a non-target amino acid. In some illustrative embodiments of pre-labeled protein standard sets, one or more selectively labeled protein standards of the set comprises a naturally-occurring protein, or a fragment thereof, that is labeled on a first (target) amino acid and that lacks a second (non-target) amino acid. In some illustrative embodiments, a selectively labeled protein standard of a pre-labeled protein standard set is labeled on a first amino acid, and comprises one or more copies of an amino acid sequence of a naturally-occurring protein, or a portion thereof, that lacks a second amino acid.
[0016]In embodiments in which a pre-labeled protein standard comprises an amino acid sequence having homology to an amino acid sequence of a naturally-occurring protein, in which the sequence is depleted in or deficient in a non-target amino acid, a selectively labeled protein of a pre-labeled protein standard set can have one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more than twenty copies of an amino acid sequence homologous to an amino acid sequence of a naturally-occurring protein.
[0017]In some preferred embodiments, a pre-labeled protein standard set can include two or more selectively labeled proteins, in which the two or more selectively labeled proteins comprise different numbers of copies of an amino acid sequence having homology to an amino acid sequence of a naturally-occurring protein that is depleted in or deficient in a non-target amino acid.
[0018]In certain embodiments, a selectively labeled protein comprises one or more copies of an amino acid sequence that is not homologous to a sequence of a naturally-occurring protein, in which the amino acid sequence is depleted in or deficient in a non-target amino acid. For example, in some embodiments of pre-labeled protein standard sets, one or more selectively labeled protein standards of the set comprises one or more copies of an amino acid sequence that is not known to have homology to a naturally-occurring protein and the one or more selectively labeled proteins is labeled on a first, or target, amino acid and is depleted in a second (non-target) amino acid. In some illustrative embodiments, a selectively labeled protein standard of a pre-labeled protein standard set comprises one or more copies of an amino acid sequence not known to occur in a naturally-occurring protein, that lacks a non-target amino acid. For example, a protein not related to a known naturally-occurring protein can be designed to be depleted in, preferably deficient in, a non-target amino acid and synthesized recombinantly or by chemical peptide synthesis.
[0019]In embodiments in which a pre-labeled protein standard comprises an amino acid sequence not derived from a naturally-occurring protein, in which the sequence is depleted in or deficient in a non-target amino acid, a selectively labeled protein of a pre-labeled protein standard set can have one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more copies of an amino acid sequence not derived from the naturally-occurring protein.
[0020]In some preferred embodiments of the invention, a pre-labeled protein standard set can include two or more selectively labeled proteins, in which each of the two or more selectively labeled proteins comprise different numbers of copies of a sequence not homologous to a naturally-occurring protein in which the sequence is depleted in or deficient in a non-target amino acid.
[0021]In another aspect of the invention, the invention provides a pre-labeled protein standard set comprising a plurality of labeled proteins, in which one or more of the proteins of the plurality is selectively labeled, in which a selectively labeled protein comprises a labeling compound on a first amino acid, and the selectively labeled protein includes an amino acid sequence having homology to an amino acid sequence of a naturally-occurring protein, in which the sequence has a reduced number of residues of a second amino acid that reacts with a labeling compound when compared with the wild type amino acid sequence of the naturally-occurring protein. In some illustrative embodiments of these aspects of the invention, a selectively labeled protein standard is a protein that is labeled on a target amino acid and comprises one or more copies of an amino acid sequence that is homologous to a sequence of a naturally-occurring protein, in which the sequence having homology to an amino acid sequence of a naturally-occurring protein sequence lacks a non-target amino acid. The invention thus includes sets of pre-labeled protein standards that comprise a plurality of labeled proteins, in which one or more of the labeled proteins is a selectively labeled protein that comprises one or more copies of an amino acid sequence that is at least 60%, at least 70%, at least 80% or at least 90% homologous to at least 20, 30, 40, 50 or more contiguous amino acids of a naturally-occurring protein, in which the homologous sequence lacks residues of a second amino acid capable of reacting with the labeling compound, and comprises a labeling compound conjugated to a first amino acid.
[0022]In some preferred embodiments of the invention, a pre-labeled protein standard set can include two or more selectively labeled proteins, in which the two or more selectively labeled proteins each comprise a different number of copies of an amino acid sequence having homology to an amino acid sequence of a naturally-occurring protein. In some preferred embodiments of the invention, a pre-labeled protein standard set can include two or more selectively labeled proteins, in which the two or more proteins each comprise a different number of copies of an amino acid sequence homologous to an amino acid sequence of a nucleotide-disulfide reductase. In some illustrative examples, selectively labeled proteins of a pre-labeled protein standard include different numbers of copies of an amino acid sequence homologous to at least a portion of a thioredoxin.
[0023]In embodiments in which a pre-labeled protein standard comprises a sequence derived from a naturally-occurring protein, in which the sequence has a reduced number of residues of a nontarget amino acid relative to the naturally-occurring protein sequence, a selectively labeled protein of a pre-labeled protein standard set can have one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more copies of an amino acid sequence derived from a naturally-occurring protein. A set of pre-labeled protein standards can comprise two or more labeled proteins, in which the two or more proteins comprise different numbers of copies of a sequence derived from a naturally-occurring protein, in which the number of residues of a non-target amino acid have been reduced relative to the naturally-occurring protein sequence.
[0024]One aspect of the invention is a protein selectively labeled on lysine. The invention includes protein standard sets that comprise one or more proteins selectively labeled on lysine and depleted in cysteine. In some embodiments, a protein selectively labeled on lysine lacks cysteine residues. The invention includes pre-labeled protein standard sets that comprise a plurality of labeled proteins, in which one or more of the labeled proteins is depleted in cysteine residues and comprises a labeling compound conjugated to one or more lysine residues. The protein(s) selectively labeled on lysine can comprise an amino acid sequence that is not homologous to a known amino acid sequence of a naturally-occurring protein, or can be an amino acid sequence that has homology to the sequence of a naturally-occurring protein
[0025]In some embodiments, a protein standard selectively labeled on lysine comprises one or more copies of an amino acid sequence having homology to the amino acid sequence of a naturally-occurring protein, in which the amino acid sequence homologous to the sequence of a naturally-occurring protein has a reduced number of cysteine residues relative to the sequence of the naturally-occurring protein. The invention includes a set of pre-labeled protein standards that comprise a plurality of labeled proteins, in which one or more of the labeled proteins comprises one or more copies of an amino acid sequence homologous to an amino acid sequence of a naturally-occurring protein, in which the homologous amino acid sequence has a reduced number of cysteine residues relative to the sequence of the naturally-occurring protein.
[0026]In one embodiment, a protein selectively labeled on lysine comprises two or more copies of an amino acid sequence having 60%, 70%, 80% or greater homology to at least 20, 30, 40, or 50 amino acids of a naturally-occurring protein sequence in which the homologous amino acid sequence of the selectively labeled protein lacks cysteine. In one embodiment, a lysine-labeled protein comprises two or more copies of an amino acid sequence derived from a naturally-occurring protein sequence, in which all of the cysteine residues of the naturally-occurring protein sequence have been removed or changed to an amino acid other than cysteine. The invention also includes nucleic acid constructs that encode proteins that comprise two or more copies of an amino acid sequence derived from the sequence of a naturally-occurring protein, in which all of the cysteine codons have been deleted or changed to non-cysteine codons.
[0027]One aspect of the invention is a protein labeled on cysteine. The invention includes protein standard sets that comprise one or more proteins selectively labeled on cysteine and depleted in lysine. In some embodiments, a protein selectively labeled on cysteine lacks lysine residues. The invention includes pre-labeled protein standard sets that comprise a plurality of labeled proteins, in which one or more of the labeled proteins is depleted in lysine residues and comprises a labeling compound conjugated to one or more cysteine residues. The protein(s) selectively labeled on cysteine can comprise an amino acid sequence that is not homologous to a known amino acid sequence of a naturally-occurring protein, or can be an amino acid sequence that has homology to the sequence of a naturally-occurring protein.
[0028]In some embodiments, a protein standard selectively labeled on cysteine comprises one or more copies of an amino acid sequence having homology to an amino acid sequence of a naturally-occurring protein, in which the amino acid sequence homologous to a sequence of a naturally-occurring protein has a reduced number of lysine residues relative to the sequence of the naturally-occurring protein. The invention includes a set of pre-labeled protein standards that comprise a plurality of labeled proteins, in which one or more of the labeled proteins comprises one or more copies of an amino acid sequence homologous to an amino acid sequence of a naturally-occurring protein, in which the homologous amino acid sequence has a reduced number of lysine residues relative to the sequence of the naturally-occurring protein.
[0029]In one embodiment, a protein selectively labeled on cysteine comprises two or more copies of an amino acid sequence having homology to an amino acid sequence of a naturally-occurring protein in which the derived amino acid sequence lacks lysine. In one embodiment, a cysteine-labeled protein comprises two or more copies of an amino acid sequence homologous to a naturally-occurring protein sequence, in which all of the lysine residues of the naturally-occurring protein sequence have been removed or changed to an amino acid other than lysine. The invention also includes nucleic acid constructs that encode proteins that comprise two or more copies of an amino acid sequence homologous to an amino acid sequence of a naturally-occurring protein, in which all of the lysine codons have been deleted or changed to non-lysine codons.
[0030]In some embodiments, a protein of a pre-labeled protein standard set that is selectively labeled on cysteine comprises an amino acid sequence homologous to an amino acid sequence of an nucleotide-disulfide oxidoreductase, such as a lipoamide dehydrogenase, a glutathione reductase, or a thioredoxin that is depleted in lysine residues. In some preferred embodiments, an amino acid sequence is homologous to an amino acid sequence of a thioredoxin, for example, homologous to a truncated thioredoxin sequence. In some preferred embodiments, an amino acid sequence homologous to an amino acid sequence of a thioredoxin differs from the naturally-occurring thioredoxin sequence by lacking lysine residues. In some preferred embodiments, a selectively labeled pre-labeled protein standard is devoid of lysine residues and is labeled on one or more cysteine residues, and comprises one or more copies of an amino acid sequence homologous to at least a portion of a thioredoxin.
[0031]The invention in some aspects provides pre-labeled protein standard sets that comprise a plurality of labeled proteins, in which two or more of the labeled proteins comprise a labeling compound conjugated to a first amino acid, and the ratios of the number of residues of the first amino acid to molecular weight for the two or more labeled proteins are within 10%, 5%, 2.5%, or 1% of one another. In some preferred embodiments, the proteins having ratios of first amino acid to molecular weight within 10%, 5%, 2.5%, or 1% of one another are selectively labeled on a first amino acid. In some exemplary embodiments, pre-labeled protein standard sets of the invention comprise a plurality of labeled proteins, in which two or more of the labeled proteins comprise a labeling compound on a first amino acid and lack residues of a second amino acid, in which the ratios of the number of residues of the first amino acid to molecular weight of the two or more selectively labeled proteins are within 10%, 5%, 2.5%, or 1% of one another.
[0032]In some preferred embodiments of a pre-labeled protein standard set, at least two proteins comprising a labeling compound on a first amino acid have between one and ten residues of a first amino acid per 10 kDa, such as between two and seven residues of a first amino acid, such as between three and five residues of a first amino acid, such as between 3.5 and 4.5 residues of a first amino acid per 10 kDa. In some preferred embodiments of a pre-labeled protein standard set, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten proteins labeled on a first amino acid have between one and ten residues of a first amino acid per 10 kDa, such as between two and seven residues of a first amino acid, such as between three and five residues of a first amino acid, such as between 3.5 and 4.5 residues of a first amino acid per 10 kDa.
[0033]In some aspects of the invention, a pre-labeled protein standard set can include one or more proteins labeled on a first amino acid that include one or more copies of an amino acid sequence derived from a naturally-occurring protein, in which the amino acid sequence comprises one or more amino acid changes that alter the number or spacing of a first amino acid targeted for labeling.
[0034]The selectively labeled proteins provided in some preferred embodiments of aspects of the invention do not differ substantially in their migration in denaturing acrylamide electrophoresis gels from the migration of the same proteins in unlabeled form. In some preferred embodiments, the selectively labeled proteins provided in preferred embodiments do not differ by more than 10%, more than 7%, or more than 5% in their migration in denaturing acrylamide electrophoresis gels from the migration of the same proteins in unlabeled form. In some preferred embodiments, the selectively labeled proteins having a molecular weight of greater than 10 kDa or greater do not differ by more than 5% in their migration in denaturing acrylamide electrophoresis gels from the migration of the same proteins in unlabeled form.
[0035]The proteins of a pre-labeled protein standard set provided in some preferred embodiments of aspects of the invention, when electrophoresed on a denaturing polyacrylamide gel, produce bands with widths that do not differ by more than two-fold between different proteins of the set that have molecular weights of 10 kDa or greater. In some preferred embodiments, the labeled proteins of a pre-labeled protein standard set having molecular weights between 20 kDa and 100 kDa produce visually detectable bands on electrophoresis gels having widths that do not differ by more than 50%. In some preferred embodiments, the widths of visually detectable bands produced by at least five pre-labeled proteins of a standard set do not differ by more than 30%.
[0036]Pre-labeled protein standard sets disclosed herein can include two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or more labeled proteins, in which one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or more of the labeled proteins is selectively labeled on a first amino acid. In preferred embodiments of the invention, at least two different proteins pre-labeled protein standard set are labeled with different labeling compounds, preferably two different dyes. Where a pre-labeled protein standard set includes two or more, three or more, four or more, or five or more labeled proteins, a pre-labeled protein standard can include different proteins that are labeled with two or more, three or more, four or more, or five or more different dyes.
[0037]A pre-labeled protein standard set of the invention in preferred embodiments spans a molecular weight range of from about 1 kDa to about 10 kDa, from about 5 kDa to about 50 kDa, from about 100 kDa to about 500 kDa, from about 10 kDa or less to about 100 kDa or greater, or from about 10 kDa or less to about 150 kDa or greater, or from about 5 kDa or less to about 150 kDa or greater, or from about 10 kDa or less to about 200 kDa or greater, or from about 5 kDa or less to about 200 kDa or greater, or from about 10 kDa or less to about 250 kDa or greater, or from about 5 kDa or less to about 250 kDa or greater.
[0038]In some embodiments, the invention provides pre-labeled molecular weight standard sets in which three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or more of the labeled proteins of the set differ in size from one another by molecular weight increments that are multiples of 5 kDa, 10 kDa, 20 kDa, or 50 kDa. In some illustrative embodiments, at least five, six, seven, eight, nine, or ten molecular weight markers can differ in size by increments that are multiples of 5 kDa. In some illustrative embodiments, at least five, six, seven, eight, nine, or ten molecular weight markers can differ in size by increments that are multiples of 10 kDa. In some preferred embodiments, the two or more labeled proteins are comprise a labeling compound bound to a first amino acid and comprise one or more copies of an amino acid sequence of or derived from an amino acid sequence of a naturally-occurring protein, in which the amino acid sequence of or homologous to an amino acid sequence of a naturally-occurring protein lacks residues of a second amino acid that can react with the labeling compound.
[0039]The invention also includes a set of pre-labeled protein standards as in any of the previous embodiments, in which the plurality of labeled proteins are provided in one or more solutions. A solution can include one or more buffers, reducing agents, chelators, alcohols, detergents, or dyes.
[0040]In another aspect, the invention provides methods of labeling proteins that include attaching a label to one or more lysine residues to a protein that lacks cysteine residues. The method includes: adding a labeling compound to a protein that lacks cysteine residues under conditions that allow conjugation of the dye with lysine. In these methods, a labeling compound comprises at least one amino-reactive group.
[0041]In a further aspect, the invention provides methods of labeling proteins that include attaching a label to one or more cysteine residues to a protein that lacks lysine residues. The method includes: reducing cystines of a protein that lacks lysine residues and adding a labeling compound to the protein under conditions that allow conjugation of the dye with cysteine. In these methods, a labeling compound has at least one sulfhydryl-reactive group.
[0042]In a further aspect, methods are provided for determining the molecular weight of a sample protein using a pre-labeled protein standard set provided herein. The method includes electrophoresing a sample that includes one or more proteins in a first lane of a gel and electrophoresing a pre-labeled protein standard set that comprises at least two labeled proteins that are selectively labeled on a first amino acid in a second lane of the gel, determining the migration distance of at least two of the two or more labeled proteins of the standard, determining the migration distance of at least one of the one or more sample proteins, and calculating the molecular weight of the at least one sample protein based on the migration distance and molecular weights of the at least two labeled proteins of the standard. The method can be performed using curve-fitting or point-to-point calibration based on the migration of the at least two labeled standards or by calibration of protein standard migration normalized to dye front migration.
[0043]The invention also includes kits that include the described pre-labeled protein standard sets, and further comprise one or more of one or more buffers, loading dyes, reducing agents, unlabeled protein standards, blotting membranes, pre-cast gels, or electrophoresis buffers. The components of the kit can in one or more containers, and two or more of the components of the kit can be provided in a common package (such as, for example, a box, rack, or jar). The kit can also include instructions for use, or instructions for accessing protocols for use via the internet.
[0044]The set of pre-labeled protein standards of the kit can be provided as lyophilized solids, or in solution in liquid or frozen form. A solution comprising one or more labeled protein standards of a set can include one or more buffers, reducing agents, chelators, alcohols, detergents, or dyes. The set of pre-labeled protein standards of the kit can include at five, six, seven, eight, nine, ten, eleven, twelve, or more labeled protein standards that are provided as one or more mixtures of two or more labeled standards. In some embodiments, all of the proteins of a pre-labeled protein standard set are provided in a single mixture (which can be provided in one or more aliquots) in a kit. The proteins of a pre-labeled protein standard set provided in a kit preferably span a molecular weight range of from 10 kDa or less to 100 kDa or more, and can span a molecular weight range of from 5 kDa or less to 250 kDa or more.
[0045]In yet another aspect, the invention provides methods of providing a set of pre-labeled protein standards to a customer, in which the set of pre-labeled protein standards includes any of the pre-labeled standard sets and kits disclosed herein. In one embodiment, the method includes: providing a pre-labeled protein standard set to a customer, in which at least one of the labeled proteins of the standard set is selectively labeled on a first amino acid, in exchange for revenue.
BRIEF DESCRIPTION OF THE DRAWINGS
[0046]FIG. 1A) depicts on line 2 the nucleic acid sequence of a truncated E. coli bacterial thioredoxin ORF (SEQ ID NO:9) with a C-terminal his tag, aligned with the a modified truncated E. coli bacterial thioredoxin ORF same sequence in which all of the lysine codons have been mutated to arginine codons and two cysteines have been added, and having a C-terminal his tag (SEQ ID NO:10) on line 1. B) depicts the translated amino acid sequence of truncated E. coli bacterial thioredoxin having a C-terminal his tag on line 2 (SEQ ID NO:11) aligned with the same sequence in which all of the lysines have been changed to arginines and two cysteines have been added on line 1 (SEQ ID NO:12).
[0047]FIG. 2 A) is a diagram of a nucleic acid construct (BH6mer ORF) having six copies of a truncated thioredoxin sequence lacking lysine separated by unique restriction sites. B) provides the nucleic acid sequence of BH6mer ORF (SEQ ID NO:13).
[0048]FIG. 3 A) shows a map of the pTrc BH 60 kDa "No Lysine" construct. B) provides the amino acid sequence of the pTrc BH 60 kDa expression product (SEQ ID NO:14)
[0049]FIG. 4 A) shows a map of the pTrc BH 30 kDa "No Lysine" construct. B) provides the amino acid sequence of the pTrc BH 30 kDa expression product (SEQ ID NO:15).
[0050]FIG. 5 A) shows a map of the pTrc BH 40 kDa "No Lysine" construct. B) provides the amino acid sequence of the pTrc BH 40 kDa expression product (SEQ ID NO:16).
[0051]FIG. 6 A) shows a map of the pTrc BH 50 kDa "No Lysine" construct. B) provides the amino acid sequence of the pTrc BH 50 kDa expression product (SEQ ID NO:17).
[0052]FIG. 7 provides the nucleic acid sequence of the "No Lysine" 50 kDa ORF insert (SEQ ID NO:37) generated from pTrc BH 60 kDa.
[0053]FIG. 8 A) shows a map of pTrc 110 kd. B) provides the deduced amino acid sequence of the expression product of pTrc 110 kd (SEQ ID NO:38).
[0054]FIG. 9 A) shows a map of pTrc 160 kd. B) provides the deduced amino acid sequence of the pTrc 160 kd expression product (SEQ ID NO:39).
[0055]FIG. 10 shows the sequence of a truncated Lac Z gene (SEQ ID NO:40) that was used to synthesize the pTrc 260 kd plasmid.
[0056]FIG. 11 A) shows a map of pTrc 260 kd. B) provides the deduced amino acid sequence of the pTrc 260 kd expression product (SEQ ID NO:41).
[0057]FIG. 12 depicts a scheme for synthesizing 8-anilino-1-naphthalenesulfonic acid-aminophenyl vinyl sulfone (8-ANS-APVS).
[0058]FIG. 13 depicts the reaction scheme for generating the vinyl sulfone form of Orange 16.
[0059]FIG. 14 A) shows a pre-labeled protein standard set of the invention electrophoresed on a 4-12% Bis-Tris gel with 1×MES running buffer. B) shows the same set of markers in unlabeled form electrophoresed on a 4-12% Bis-Tris gel with MES running buffer.
[0060]FIG. 15 A) shows a 4-12% Bis-Tris gel with 1×MES running buffer, B) shows a 4-12% Bis-Tris gel with 1×MOPS running buffer, and C) shows a 4-20% Tris-glycine gel on which a set of pre-labeled protein standards (Sharp Pre-stained Standard; lane 4) were electrophoresed alongside other commercially available pre-stained markers: 1--Precision Plus Blue (Bio-Rad); 2--Precision Plus Dual (Bio-Rad); 3--Precision Plus Kaleidoscope (Bio-Rad); 4--Sharp Pre-stained Standard (Invitrogen); 5--Rainbow (GE); 6--BenchMark® prestain (Invitrogen); 7--MultiMark (Invitrogen); 8--SeeBlue+2 (Invitrogen).
[0061]FIG. 16 A) depicts a ruler aligned with a gel on which pre-labeled protein standards of the invention were electrophoresed for determining band width of the pre-labeled standards. B) depicts a trace extracted from the gel image having peaks 2-13 corresponding to band intensity of the pre-labeled proteins.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0062]In the description that follows, a number of terms used in recombinant DNA technology and protein chemistry are utilized extensively. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided.
[0063]As used herein, the articles "a," "an" and "one" mean "at least one" or "one or more" of the object to which they refer, unless otherwise specified or made clear by the context in which they appear herein.
[0064]As used herein, the terms "about" or "approximately" when referring to any numerical value are intended to mean a value of ±10% of the stated value. For example, "about 50° C." (or "approximately 50° C.") encompasses a range of temperatures from 45° C. to 55° C., inclusive. Similarly, "about 100 mM" (or "approximately 100 mM") encompasses a range of concentrations from 90 mM to 110 mM, inclusive.
[0065]The term "label" as used herein refers to a chemical moiety or protein that is directly or indirectly detectable (e.g. due to its spectral properties, conformation or activity) when attached to a target or compound and used in the present methods. The label can be directly detectable (fluorophore, chromophore) or indirectly detectable (hapten or enzyme). Such labels include, but are not limited to, radiolabels that can be measured with radiation-counting devices; pigments, dyes or other chromophores that can be visually observed or measured with a spectrophotometer; spin labels that can be measured with a spin label analyzer; and fluorescent labels (fluorophores), where the output signal is generated by the excitation of a suitable molecular adduct and that can be visualized by excitation with light that is absorbed by the dye or can be measured with standard fluorometers or imaging systems, for example. The label can be a chemiluminescent substance, where the output signal is generated by chemical modification of the signal compound; a metal-containing substance; or an enzyme, where there occurs an enzyme-dependent secondary generation of signal, such as the formation of a colored product from a colorless substrate. The term label can also refer to a "tag" or hapten that can bind selectively to a conjugated molecule such that the conjugated molecule, when added subsequently along with a substrate, is used to generate a detectable signal. For example, one can use biotin as a tag and then use an avidin or streptavidin conjugate of horseradish peroxidate (HRP) to bind to the tag, and then use a colorimetric substrate (e.g., tetramethylbenzidine (TMB)) or a fluorogenic substrate such as Amplex Red reagent (Molecular Probes, Inc.) to detect the presence of HRP. Numerous labels are know by those of skill in the art and include, but are not limited to, particles, dyes, fluorophores, haptens, enzymes and their colorimetric, fluorogenic and chemiluminescent substrates and other labels that are described in RICHARD P. HAUGLAND, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES AND RESEARCH PRODUCTS (9th edition, CD-ROM, September 2002), supra.
[0066]The term "directly detectable" as used herein refers to the presence of a material or the signal generated from the material is immediately detectable by observation, instrumentation, or film without requiring chemical modifications or additional substances.
[0067]"Detectable by the naked eye" means the referred to entity is directly visible by a human being having normal vision without the aid of, for example, glasses that magnify or filter light or a microscope (or lens of any type that provides magnification), and without the aid of illumination of greater intensity than standard laboratory room fluorescent or incandescent lighting, or illumination with light of narrower wavelength(s) than standard laboratory room fluorescent or incandescent lighting, or illumination with wavelength(s) outside that of standard laboratory room fluorescent or incandescent lighting.
[0068]A "dye" is a visually detectable label. A dye can be, for example, a chromophore or a fluorophore. A fluorophore can be excited by visible light or non-visible light (for example, UV light).
[0069]A "chromophore" is a chemical group or compound capable of selective light absorption resulting in the coloration of the organic compound.
[0070]The term "fluorophore" as used herein refers to a composition that is inherently fluorescent or demonstrates a change in fluorescence upon binding to a biological compound or metal ion, i.e., fluorogenic. In many cases, fluorophores are also chromophores that have an observable color when they absorb light. Fluorophores may contain substitutents that alter the solubility, spectral properties or physical properties of the fluorophore. Numerous fluorophores are known to those skilled in the art and include, but are not limited to coumarin, cyanine, benzofuran, a quinoline, a quinazolinone, an indole, a benzazole, a borapolyazaindacene and xanthenes including fluoroscein, rhodamine and rhodol as well as other fluorophores described in RICHARD P. HAUGLAND, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS (9th edition, CD-ROM, September 2002).
[0071]A "textile dye" is a dye typically used to dye cloth fabrics and material for making cloth fabrics (e.g., fibers, yarn, thread), such as cloth fabrics that comprises, for example, cotton, wool, polyamide (nylon), polyester, viscose, acrylic, acetate, triacetate, etc. Textile dyes can also be used to dye materials and compounds other than fabrics and materials for making fabrics. Textile dyes are available from many commercial suppliers (for example, Burlington Chemical Co., Burlington, N.C.; Harneet Exports, Mumbai, India; Jagson Colorchem Ltd., Ahmadabed, India; Jaychem, Sanand, India; Omega Dyes, Goucestershire, UK; Dystar Textilfarben, Frankfurt, Germany; Kemtex, Chorley, UK). Nonlimiting examples of textiles dyes are Remazol dyes, Kemozol dyes, Direct dyes, Disperse dyes, Dischargeable acid dyes, Kenanthol dyes, Kenamide dyes, Cibacron dyes, azoic dyes, Dyacid dyes, Kemtex reactive dyes, Kemtex acid dyes, Kemtex Easidye acid dyes, Calcdon dyes, Cassulfon dyes, Isolan dyes, Sirius dyes, Imperon dyes, phtalogen dyes, naphtol dyes, Levafix dyes, Procion dyes, and isothiocyanate dyes.
[0072]A "pre-labeled" biomolecule is a biomolecule that includes a label prior to performing a separation or experiment with the biomolecule. For example, a pre-labeled standard is labeled prior to separation of that standard by biochemical techniques such as, but not limited to, electrophoresis (including both solution phase and gel electrophoresis), isoelectric focusing, spectrometry, or chromatography.
[0073]In the context of the present invention, "selectively labeled" means labeled predominantly on particular sites of a biomolecule. In particular, a protein that is "selectively labeled" on a [first] amino acid is a protein that has been conjugated with a labeling compound that has a reactive chemical group that is specific for the [first] amino acid, and that either has fewer than one residue per 10 kDa of one or more other (second) amino acids that can also react with the labeling compound, or has a chemical modification of one or more other (second) amino acids that can also react with the labeling compound. Selective labeling of proteins is accomplished by the use of labeling compounds having reactive chemical groups that are specific for one or more particular chemical groups present on one or more amino acids on proteins, and by reducing side-reactions of the reactive group of the dye with one or more other amino acids that are capable of reacting with the reactive group of the dye. Reducing side reactions can be by either or both of: modifying one or more chemical groups that are capable of reacting with the reactive group of the dye such that they are no longer capable of reacting with the labeling compound under the reaction conditions used to label the protein, and selecting a protein for labeling that is depleted in amino acids that have chemical groups capable of reacting with the dye used for labeling the protein.
[0074]"Amino acid" refers to the twenty naturally-occurring amino acids, as well as to derivatives of these amino acids that occur in nature or are produced outside of living organisms by chemical or enzymatic derivatization or synthesis (for example, hydroxyproline, selenomethionine, azido-labeled amino acids, etc.)
[0075]In the context of the present application, a "target amino acid" or "an amino acid targeted for labeling" is an amino acid that is used for the covalent attachment of a label, such as a dye, to a peptide or protein. "Target amino acid" refers to an amino acid species, for example lysine, by which is meant all lysine residues of a protein, and is not used to refer to a single particular lysine residue of a protein. In making labeled protein standards of the invention, a target amino acid is an amino acid whose labeling is intended; the labeling of a protein on a target amino acid is achieved by selecting a labeling compound with a reactive chemical group that reacts with the reactive chemical group on the target amino acid.
[0076]A "nontarget amino acid" is an amino acid on a protein standard that has a reactive group that is capable of reacting with a labeling compound conjugated to a target amino acid of the protein standard, but whose conjugation to a labeling compound is not desired. A "nontarget amino acid" can have the same reactive chemical group as a target amino acid or a different reactive chemical group. A non-target amino acid can have greater, less, or substantially the same affinity for a labeling compound as a target amino acid.
[0077]A protein that is "depleted in an amino acid" means that the protein has fewer than one residue of the amino acid per 10 kDa. In some preferred embodiments of the invention, a protein standard that is depleted in a non-target amino acid has no residues of a non-target amino acid (lacks a non-target amino acid).
[0078]A protein that is "deficient in an amino acid" means that the protein has no residues of the amino acid.
[0079]"Conservative amino acid substitutions" refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having acidic side chains is glutamic acid and aspartic acid; a group of amino acids having amino-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine and tryptophan; a group of amino acids having basic side chains is lysine, arginine and histidine; and a group of amino acids having sulfur-containing side chain is cysteine and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine; phenylalanine-tyrosine; lysine-arginine; alanine-valine; glutamic acid-aspartic acid; and asparagine-glutamine.
[0080]The term "reactive group" or "reactive chemical group" as used herein refers to a chemical group that is capable of reacting with another chemical group to form a covalent bond, i.e. is covalently reactive under suitable reaction conditions, and generally represents a point of attachment for another substance. The reactive group is a moiety, such as carboxylic acid or succinimidyl ester, on the compounds of the present invention that is capable of chemically reacting with a functional group on a different compound to form a covalent linkage. Reactive groups generally include without limitation nucleophiles, electrophiles and photoactivatable groups.
[0081]"Conjugated to" means covalently bound to. A molecule or chemical group that is conjugated to another molecule or chemical group is covalently bound. To conjugate [a molecule or chemical group to another molecule or chemical group] is to cause or promote a chemical reaction between the two referenced molecules or chemical groups such that they become covalently bound.
[0082]As used herein an amino acid or reactive group of an amino acid that "reacts with" a labeling compound becomes covalently bound to the labeling compound.
TABLE-US-00001 TABLE 1 Reactive Groups of Amino Acids Specific type of pKa of side chain Amino Acid Reactive Group reactive group (theoretical) Cysteine sulfhydryl 8.8-9.1 N-terminal amine Alpha amine 7.6-8.0 Lysine amine Epsilon amine 9.3-9.5 Histidine amine Imidazole 6.7-7.1 Tryptophan amine Indoyl amine -- Arginine amine Guanidino amine >12.0 C-terminal carboxyl Alpha carboxyl 2.1-2.4 Aspartic acid carboxyl Beta carboxyl 3.7-4.0 Glutamic acid carboxyl Gamma carboxyl 4.2-4.5 Tyrosine hydroxyl Phenolate 9.7-10.1 Methionine thioether -- Asparagine amidino
[0083]As used herein, "protein" means a polypeptide, or a sequence of two or more amino acids, which can be naturally-occurring or synthetic (modified amino acids, or amino acids not known in nature) linked by peptide bonds. "Peptide" specifically refers to polypeptides of less than 10 kDa. As used herein, the term "protein" encompasses peptides.
[0084]"Naturally-occurring" refers to the fact that an object having the same composition can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism, including viruses, that can be isolated from a source in nature, and that has not been intentionally modified in the laboratory is naturally-occurring.
[0085]A nucleic acid (or nucleotide) or protein (or amino acid) sequence that is "derived from" another nucleic acid (or nucleotide) or protein (or amino acid) sequence is either the same as at least a portion of the sequence it is derived from, or highly homologous to at least a portion of the sequence it is derived from, having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity with the sequence of the protein from which it is derived. An amino acid sequence derived from the sequence of a naturally-occurring protein can be referred to as a "naturally-occurring protein-derived amino acid sequence" or, simply, "a derived [amino acid] sequence". A nucleic acid sequence derived from the sequence of a naturally-occurring nucleic acid can be referred to as a "naturally-occurring nucleic acid-derived nucleic acid sequence" or, simply, "a derived [nucleic acid] sequence".
[0086]"Homologous" means that a protein peptide, or amino acid sequence has at least 65%, at least 70% amino acid sequence identity, at least 80% amino acid sequence identity, preferably 90% amino acid sequence identity, and more preferably at least 95% amino acid sequence identity with amino acid sequence referred to. The sequence having homology with another amino acid sequence has at least six amino acids, preferably at least 10 amino acids, and more preferably at least twenty, at least thirty, or at least forty contiguous amino acids of the protein, peptide, or amino acid sequence referred to.
[0087]A "variant" of a wild-type protein or peptide sequence is a sequence having at least 70%, preferably at least 80%, at least 90%, at least 95%, or at least 99% sequence identity with at least 20 contiguous amino acids of the wild-type protein.
[0088]"Recombinant methods" are methods that include the manufacture of or use of recombinant nucleic acids (nucleic acids that have been recombined to generate nucleic acid molecules that are structurally different from the analogous nucleic acid molecule(s) found in nature). Recombinant methods can employ, for example, restriction enzymes, exonucleases, endonucleases, polymerases, ligases, recombination enzymes, methylases, kinases, phosphatases, topoisomerases, etc. to generate chimeric nucleic acid molecules, generate nucleotide sequence changes, or add or delete nucleic acids to a nucleic acid sequence. Recombinant methods include methods that combine a nucleic acid molecule directly or indirectly isolated from an organism with one or more nucleic acid sequences from another source. The sequences from another source can be any nucleic acid sequences, for example, gene expression control sequences (for example, promoter sequences, transcriptional enhancer sequences, sequence that bind inducers or promoters of transcription, transcription termination sequences, translational regulation sequences, internal ribosome entry sites (IRES's), splice sites, poly A addition sequences, poly A sequences, etc.), a vector, protein-encoding sequences, etc. The nucleic acid sequences from a source other than the source of the nucleic acid molecule directly or indirectly isolated from an organism can be nucleic acid sequences from or within the genome of a different organism. Nucleic acid sequences in the genome can be chromosomal or extra-chromosomal (for example, the nucleic acid sequences can be episomal or of an organelle genome). Recombinant methods also includes methods of introducing nucleic acids into cells, including transformation, viral transfection, etc. to establish recombinant nucleic acid molecules in cells. "Recombinant methods" also includes the synthesis and isolation of products of nucleic acid constructs, such as recombinant RNA molecules and recombinant proteins. "Recombinant methods" is used interchangeably with "genetic engineering" and "recombinant [DNA] technology".
[0089]A "recombinant protein" is a protein made from a recombinant nucleic acid molecule or construct. A recombinant protein can be made in cells harboring a recombinant nucleic acid construct, which can be cells of an organism or cultured prokaryotic or eukaryotic cells, or can made in vitro using, for example, in vitro transcription and/or translation systems.
[0090]"Do not differ substantially" or "substantially the same" means that the referenced compositions or components differ by less than 10% of the larger of the compared values.
[0091]The term "purified" as used herein refers to a preparation of a protein that is essentially free from contaminating proteins that normally would be present in association with the protein, e.g., in a cellular mixture or milieu in which the protein or complex is found endogenously such as serum proteins or cellular lysate.
[0092]"Substantially purified" refers to the state of a species or activity that is the predominant species or activity present (for example on a molar basis it is more abundant than any other individual species or activities in the composition) and preferably a substantially purified fraction is a composition wherein the object species or activity comprises at least about 50 percent (on a molar, weight or activity basis) of all macromolecules or activities present. Generally, a substantially pure composition will comprise more than about 80 percent of all macromolecular species or activities present in a composition, more preferably more than about 85%, 90%, or 95%.
[0093]The term "sample" as used herein refers to any material that may contain a biomolecule or an analyte for detection or quantification. The biomolecule or analyte may include a reactive group, e.g., a group through which a compound of the invention can be conjugated to the analyte. The sample may also include diluents, buffers, detergents, and contaminating species, debris and the like that are found mixed with the target. Illustrative biological examples include urine, sera, blood plasma, total blood, saliva, tear fluid, cerebrospinal fluid, secretory fluids from nipples and the like. Also included are solid, gel or sol substances such as mucus, body tissues, cells and the like suspended or dissolved in liquid materials such as buffers, salts, alcohols, extractants, lipids, solvents, detergents, reducing agents, chelators, anti-coagulants, preservatives, anti-microbial agents, and the like. A sample can include one or more partially or substantially purified biomolecules or analyte. A sample can be a live cell, a biological fluid that comprises endogenous host cell proteins, nucleic acid polymers, nucleotides, oligonucleotides, peptides and buffer solutions. The sample can be in an aqueous solution, a viable cell culture or immobilized on a solid or semi solid surface such as a polyacrylamide gel, membrane blot or on a microarray.
[0094]Two or more proteins "have electrophoretic separation characteristics that are substantially the same" or "do not differ substantially in their migration in acrylamide electrophoresis gels" when the molecular weights calculated for the two or more referenced proteins by their migration distance on a gel, such as a polyacrylamide gel, are within 10%, preferably within 7% or within 5%. The calculated molecular weights of the proteins can be performed by curve-fitting of molecular weight to migration distances or point-to-point calculation.
Pre-Labeled Protein Standard Sets with Proteins Selectively Labeled on a First Amino Acid
[0095]The invention provides pre-labeled protein standard sets comprising a plurality of labeled proteins, in which one or more of the labeled proteins is selectively labeled on a first amino acid. A protein selectively labeled on a first amino acid is a protein that comprises a labeling compound conjugated to one or more residues of a first amino acid and either: a) is depleted in residues of a second amino acid that reacts with a labeling compound; or b) comprises one or more copies of an amino acid sequence derived from the amino acid sequence of a naturally-occurring protein, in which the amino acid sequence derived from the amino acid sequence of a naturally-occurring protein has a reduced number of residues of a second amino acid capable of reacting with the labeling compound relative to the wild-type amino acid sequence of the naturally occurring protein.
[0096]A protein that is depleted in residues of a second amino acid is a protein that has fewer than one residue of the second amino acid per 10 kDa. A protein that is depleted in residues of a second amino acid can have no residues of a second amino acid.
[0097]In the context of the present invention, a first amino acid is an amino acid whose labeling is desired, and whose labeling is targeted by the choice of reactive group on a labeling compound. A first amino acid is referred to herein as a "target amino acid".
[0098]In the context of the present invention, a second, or non-target, amino acid is an amino acid whose labeling is not desired, but that has a reactive chemical group that, under conditions used to label the protein on a first amino acid, reacts with the labeling compound that is used to label the protein. In some embodiments, a non-target amino acid has the same reactive group as the target amino acid. In some embodiments, a non-target amino acid has a different reactive group from the target amino acid. The present invention seeks to reduce labeling of non-target amino acids by reducing their occurrence in a protein used as a pre-labeled protein standard.
[0099]One or more proteins of a set of labeled protein standards can be selectively labeled, for example, on the sulfhydryl group of cysteine, on the primary amine of an N-terminal amino acid and/or the primary amine of lysine, on the secondary amine of the imidazoyl group of histidine or the indole ring of tryptophan, on the carboxyl groups of the C-terminal amino acid or of aspartate or glutamate, on the thioether of methionine, on the phenolate of tyrosine, or on the amidino group of asparagine. Any of the amino acids: cysteine, lysine, histidine, tryptophan, aspartate, glutamate, methionine, tyrosine, or asparagines can be target amino acids to which a labeling compound can be conjugated. Any of the amino acids cysteine, lysine, histidine, tryptophan, aspartate, glutamate, methionine, tyrosine, or asparagine can also be a non-target amino acid whose interaction with a labeling compound is sought to be reduced or eliminated when a protein is labeled on a first amino acid.
[0100]For example, in some exemplary embodiments, cysteine can be a target amino acid and one or more of lysine histidine, or tryptophan can be a non-target amino acid. In other exemplary embodiments, lysine can be a target amino acid and one or more of cysteine, histidine, or tryptophan can be a non-target amino acid.
[0101]A selectively labeled protein can have more than one target amino acid. For example, both glutamate and aspartate can be target amino acids. A selectively labeled protein can have more than one non-target amino acid. For example, lysine can be a target amino acid, and two or more of cysteine, arginine, histidine, and tryptophan can be non-target amino acids.
[0102]In yet other embodiments, the first amino acid is histidine and the second amino acid is one or more of cysteine, lysine, or tryptophan. In other embodiments, the first amino acid is tryptophan and the second amino acid is one or more of cysteine, lysine, histidine, or asparagines. In further embodiments, the first amino acid is asparagine and the second amino acid is one or more or cysteine, lysine, histidine, or tryptophan. In additional embodiments, the first amino acid is tyrosine and the second amino acid is one or more of cysteine, lysine, histidine, or tryptophan. The first amino acid can in yet further embodiments be methionine and the second amino acid can be one or more of cysteine, lysine, histidine, tyrosine, or tryptophan.
[0103]Selectively labeled protein standards of the invention comprise a labeling compound on a first amino acid (a target amino acid) and are depleted in a second amino acid (a non-target amino acid), or comprise a labeling compound on a first amino acid (a target amino acid) and comprise an amino acid sequence having homology to an amino acid sequence of a naturally-occurring protein, in which the derived sequence has fewer residues of the second amino acid with respect to the wild-type sequence of the naturally occurring protein. In targeting an amino acid for labeling, a labeling compound is selected that has a reactive group that specifically reacts with the reactive group of the target amino acid to form a covalent bond, thereby forming a labeling compound-protein conjugate, or labeled protein. Preferably, a labeling compound used to label a protein standard has a high specificity for the reactive group of the target amino acid. Labeling compounds can be selected based on their reactive groups, or can be modified, using methods known in the art, to have reactive groups with high specificity for a target amino acid. Preferably, reaction conditions that optimize the reaction of the reactive chemical groups of the labeling compound and target amino acid are used for conjugating a selected label to the target amino acid.
[0104]A second amino acid, or non-target amino acid, is an amino acid that is capable of reacting with a labeling compound used to label a target amino acid of a protein under reaction condition used to conjugate the labeling compound to a target amino acid, but whose conjugation with a labeling compound is not desired. A non-target amino acid can be capable of reacting with a label used to label a target amino acid with substantially the same efficiency as the target amino acid, with reduced efficiency with respect to the reaction of the target amino acid with the label, or with greater efficiency with respect to the reaction of the target amino acid with the label. In certain illustrative examples, the non-target amino acid is capable of reacting with the label more efficiently than any other amino acid in the protein, except for the first amino acid.
[0105]In selecting one or more target amino acids and minimizing labeling of one or more non-target amino acids for labeling a protein standard, the reactivities of the groups present on amino acid side chains are taken into account. For example, the side chains of several amino acids include chemical groups that can act as nucleophiles in chemical conjugation reactions. Examples of such reactive chemical groups of amino acids include, without limitation, the sulfhydryl group of cysteine, the alpha amino group of N-terminal amino acids, the epsilon amino group of lysine, the imidazole amino group of histidine, the indoyl amino group of tryptophan, the guanidino group of arginine, the carboxyl group of the C-terminal amino acid of a protein, the carboxyl group of glutamic acid, the carboxyl group of aspartic acid, the phenolate of tyrosine, the thioether of methionine, and the amidino group of arginine. Reactions of these groups with a nucleophile-interacting group of a label will be more or less efficient depending on factors that include but are not limited to the reactive group of the label, the strength of the nucleophile group of the amino acid, and the pH at which the reaction occurs. For example, the sulfhydryl group of cysteine is generally a stronger nucleophile than the amino groups of lysine, the N-terminus of a protein, histidine, and tryptophan, which are stronger nucleophiles than the carboxyl groups of the C-terminus of a protein, aspartic acid, and glutamic acid, and the phenolate of tyrosine. In some preferred embodiments, a target amino acid of a pre-labeled protein standard can be an amino acid such as, but not limited to, cysteine, lysine, histidine, tryptophan, aspartic acid, glutamic acid, tyrosine, arginine, methionine, an N-terminal amino acid of the protein, or a C-terminal of the protein, in which one or more amino acids that also can undergo nucleophilic addition are non-target amino acid(s) that can be depleted in a pre-labeled protein standard.
[0106]For example, an amino acid having a chemical group that behaves as a nucleophile at a pH greater than neutrality, such as, for example, cysteine, lysine, tryptophan, or histidine, can be a target amino acid and one or more of the same group of amino acids that behave as nucleophiles at a pH greater than neutrality can be a non-target amino acid that is depleted in a labeled protein standard or is present in reduced amounts (relative to the corresponding wild-type protein sequence) in a labeled protein standard. In embodiments in which at least one of lysine, histidine, or tryptophan is a target amino acid, a label preferably includes an amino-reactive group for conjugation to the standard. Examples of amino-reactive groups that can be present on a compound used to label lysine, histidine, tryptophan, or an N-terminal amino acid include, but are not limited to, isothiocyanates, isocyanates, acyl azides, N-hydroxysuccinimide (NHS) esters, haloacetyl compounds, maleimide derivatives, sulfonyl chlorides, aldehydes, ketones, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, carbodiimides, or acid anhydrides.
[0107]In one example, lysine can be a target amino acid, and one or more of, for example, cysteine, histidine, or tryptophan can be non-target amino acid(s). In some illustrative embodiments, a selectively labeled protein standard selectively labeled on lysine is depleted in or lacks residues of at least one of cysteine, histidine, or tryptophan. A protein standard selectively labeled on lysine can optionally be made by recombinant methods from a nucleic acid construct that encodes at least a portion of a sequence of a naturally-occurring protein, in which one or more cysteine, histidine, or tryptophan codons has been removed by mutation or deletion. In some preferred embodiments, a protein standard selectively labeled on lysine is made from a nucleic acid construct in which all of the codons for at least one of cysteine, histidine, or tryptophan, or any combinations thereof, have been removed by deletion or mutation. A labeled protein standard of the invention that is selectively labeled on lysine can lack residues of one or more non-target amino acids and can have one or more additional non-target amino acids that are chemically modified such that they do not react with the labeling compound conjugated to the first amino acid.
[0108]In another example, cysteine can be a target amino acid, and one or more of lysine, tryptophan, or histidine, can be non-target amino acid(s). In these embodiments, preferably at least lysine is a non-target amino acid, since the reactivity of the primary amine of lysine is greater than that of the indoyl or imidazole amines of tryptophan or histidine, and thus lysine contributes more significantly to side reactions when conjugating a compound to cysteine. For example, cysteine can be a target amino acid of a pre-labeled protein standard where the labeling compound attached to the pre-labeled standard is a labeling compound that, prior to conjugation with the protein, comprised a reactive chemical group that reacts with the sulfhydryl group of cysteine, such as but not limited to: vinyl sulfone, iodoacetamide, maleimide, disulfides, mercurial compounds, haloacetyl compounds, and iodoacetic acid. In one example, a selectively labeled protein standard has a labeling compound conjugated to at least one cysteine residue and lacks residues of one or more of lysine, histidine, or tryptophan. In some preferred embodiments, a protein standard selectively labeled on cysteine is depleted in or has an amino acid sequence with a reduced number of residues of at least lysine relative to the corresponding wild-type amino acid sequence. A protein standard selectively labeled on cysteine can optionally be made by recombinant methods from a nucleic acid construct that encodes at least a portion of a sequence of a naturally-occurring protein, in which one or more lysine, histidine, or tryptophan codons has been removed. In some preferred embodiments, a protein standard selectively labeled on cysteine is made from a nucleic acid construct in which all of the codons for at least one of lysine, histidine, or tryptophan have been removed by deletion or mutation. A labeled protein standard of the invention that is selectively labeled on cysteine can lack one or more non-target amino acids and can have one or more additional non-target amino acids that are chemically modified.
[0109]In another example, an amino acid having a chemical group that behaves as a nucleophile at a pH lower than neutrality, for example, aspartate or glutamate, can be a target amino acid and one or more other amino acids that behaves as a nucleophile at a pH less than neutrality can be a non-target amino acid that is not present in a labeled protein standard or modified in a labeled protein standard. In one example, aspartate can be a target amino acid, and glutamate can be a non-target amino acid. In another example, glutamate can be a target amino acid, and aspartate can be a non-target amino acid. A labeling compound for glutamate or aspartate can include a carboxyl-reactive group, such as but not limited to, a diazoalkane, a diazoacetyl, a carbonyldiimidazole, or a carbodiimide.
[0110]Tyrosine can also be a target amino acid, in which a reactive chemical group on a label to be conjugated to the protein standard is, for example, a sulfonyl fluoride or iodoacetamide. Another potential target amino acid is methionine, in which a reactive chemical group on a compound used to label the protein standard is, for example, a haloacetate, a haloacetyl, or an aryl halide. Arginine can be a target amino acid, in which a chemical group on a compound used to label the protein is an oxalyl group.
[0111]In any of these examples an N-terminal amino acid, which can be labeled on the N-terminal amino group, can be a target amino acid or a non-target amino acid.
[0112]More than one amino acid can be targeted for selectively labeling a protein. For example, the N-terminal amino acid of a protein as well as lysine can be target amino acids, where a labeling compound conjugated to the selectively labeled protein includes a reactive chemical group that reacts with primary amines. In another example, glutamate, aspartate, and the C-terminal amino acid of a protein can be target amino acids, where a dye conjugated to the selectively labeled protein includes a reactive chemical group that reacts with carboxylates.
[0113]Methods for conjugating a label to particular amino acids of a protein, for example, the amino group of lysine residues, the N-terminus of the protein, histidine, and/or tryptophan; the sulfhydryl group of cysteine; the carboxyl group of aspartate and glutamate; as well as the thioether of methionine, and the phenolate of tyrosine are well known in the art (see, for example, Hermanson, Bioconjugate Techniques, Academic Press, San Diego (1996); Wong, Chemistry of Protein Conjugation and Cross-Linking, CRC Press, Boca Raton, 1993; Haugland, MOLECULAR PROBES HANDBOOK, available at www.invitrogen.com, (2002)). In general, methods for conjugation of a labeling compound to an amino acid residue of a protein comprise: [0114]a) combining a protein that comprises a first amino acid that comprises a first reactive group with a labeling compound that comprises a second reactive group that reacts with the first reactive group, to form a protein-labeling compound mixture; and, [0115]b) incubating the protein-labeling compound mixture for a sufficient amount of time for the labeling compound to form a covalent bond with first reactive group of the first amino acid, wherein a labeled protein standard is formed.In some preferred embodiments in which a first amino acid is cysteine, and the reactive group of cysteine is a sulfhydryl group, the method preferably also comprises: [0116]c) prior to a), combining a protein that comprises one or more cysteine residues with a reducing agent; and [0117]d) incubating the protein with the reducing agent for a sufficient amount of time for cysteine-cysteine bonds to be reduced.
[0118]In some aspects, the invention includes a method for making a protein standard, comprising attaching a label to one or more cysteine residues of a protein that is depleted in lysine residues. For example, the method in some embodiments includes attaching a label that includes a sulfhydryl-reactive group, such as but not limited to a vinyl sulfone, an iodoacetamide, an maleimide, a disulfide, a mercurial compound, a haloacetyl compound, or an iodoacetic acid, to a protein that is depleted in lysine residues. In some embodiments, the protein that is depleted in lysine residues comprises an amino acid sequence that has homology to at least 40 amino acids of a naturally-occurring protein, such as at least 70%, at least 80%, or at least 90% homology to at least 40 amino acids of a naturally-occurring protein, and has fewer lysine residues than the amino acid sequence of the naturally-occurring protein to which it has homology. In some embodiments, the protein that is depleted in lysine residues comprises fewer than one residue of lysine per 10 kDa. In some embodiments, the protein that is depleted in lysine residues has no lysine residues.
[0119]In some aspects, the invention includes a method for making a protein standard, comprising attaching a label to one or more lysine residues of a proteins that is depleted in cysteine residues. For example, the method in some embodiments includes attaching a label that includes an amino-reactive group, such as but not limited to an isothiocyanate, an isocyanate, an acyl azide, an N-hydroxysuccinimide (NHS) ester, a haloacetyl compound, a maleimide derivative, a sulfonyl chloride, an aldehyde, a ketone, a glyoxal, an epoxide, an oxirane, a carbonate, an aryl halide, an imidoester, a carbodiimide, or an acid anhydride, to a protein that is depleted in cysteine residues. In some embodiments, the protein that is depleted in cysteine residues comprises an amino acid sequence that has homology to at least 40 amino acids of a naturally-occurring protein, such as at least 70%, at least 80%, or at least 90% homology to at least 40 amino acids of a naturally-occurring protein, and has fewer cysteine residues than the amino acid sequence of the naturally-occurring protein to which has homology. In some embodiments, the protein that is depleted in cysteine residues comprises fewer than one residue of cysteine per 10 kDa. In some embodiments, the protein that is depleted in cysteine residues has no cysteine residues.
[0120]In preferred methods, the labeling compound is a dye. Reactive dyes and their preparation are well known in the art (Haugland, MOLECULAR PROBES HANDBOOK, supra, (2002)).
[0121]In some preferred methods of labeling cysteine residues, the reducing agent is beta-mercaptoethanol, dithiothreitol, TCEP, or TBP. Reducing agents can be used at concentrations ranging from about 0.01 millimolar to about 50 millimolar, for example, from about 0.05 micromolar to about 20 millimolar, or from about 0.1 millimolar to about 10 millimolar, or from about 0.2 mM to about 5 mM, or from about 0.5 mM to about 2 mM.
[0122]Preferably, conjugation to form a covalent bond consists of simply mixing the reactive compounds of the present invention in a suitable solvent in which both the reactive compound and the substance to be conjugated are soluble. The reaction preferably proceeds spontaneously without added reagents at a suitable temperature.
[0123]Conjugation methods can vary and can be optimized according to the purposes of the practitioner, so the following description is illustrative and not limiting to the invention. Preparation of peptide or protein conjugates typically comprises first dissolving the protein to be conjugated in aqueous buffer at about. 1-10 mg/mL at room temperature or below. Bicarbonate buffers (pH about 8.3) are especially suitable for reaction with succinimidyl esters, phosphate buffers (pH about 7.2-8) for reaction with thiol-reactive functional groups and carbonate or borate buffers (pH about 9) for reaction with isothiocyanates and dichlorotriazines. The appropriate reactive label compound is dissolved in a nonhydroxylic solvent (usually DMSO or DMF) in an amount sufficient to give a suitable degree of conjugation when added to a solution of the protein to be conjugated. The appropriate amount of compound for any protein or other component is conveniently predetermined by experimentation in which variable amounts of the compound are added to the protein, the conjugate is purified (for example, using chromatography) to separate unconjugated compound and the protein-labeling compound conjugate is tested in its desired application. It is generally preferred that the reagents be kept as concentrated as practical so as to obtain adequate rates of conjugation. An excess of labeling compound over target amino acid is typically used in the labeling reaction.
[0124]Following addition of the reactive compound to the component solution, the mixture is incubated for a suitable period. The incubation can occur at any temperature, from close to 0 degrees C. to about 90 degrees C., but typically is for about 1 hour at room temperature or above (such as up to 60 degrees C.) to several hours on ice. After incubation, the excess labeling compound is removed by gel filtration, dialysis, HPLC, precipitation, adsorption on an ion exchange or hydrophobic polymer, or other suitable means. The dye-protein conjugate can be stored or used in solution or lyophilized.
[0125]Selectivity of labeling is best obtained by selection of an appropriate reactive dye. For example, modification of thiols with a thiol-selective reagent such as a haloacetamide, vinyl sulfone, or maleimide, or modification of amines with an amine-reactive reagent such as an activated ester, acyl azide, isothiocyanate or 3,5-dichloro-2,4,6-triazine. Partial selectivity can also be obtained by careful control of the reaction conditions.
[0126]A labeling compound conjugated to a protein standard can be any type of label, but is preferably a directly detectable label, and is more preferably a dye that can be visually detected with the naked eye. Preferably, a labeling compound is a dye detectable with the naked eye such that labeled proteins can be detected in a gel immediately after, and preferably during, electrophoresis without the need for additional processing or image analysis of the gel. Preferably, a labeling compound is not an unmodified naturally-occurring amino acid.
[0127]The invention provides protein standards that behave in separation procedures substantially the same as their unlabeled counterparts; therefore the labels used in the invention are preferably of relatively low molecular weight, such as molecular weight of less than about 2 kDa, preferably less than about 1.5 kDa, more preferably less than about 1 kDa, and can be less than about 0.5 kDa. For example, the molecular weight of a labeling compound can be between about 0.1 kDa and about 1 kDa, or between about 0.2 kDa and about 1.5 kDa, or between about 0.3 kDa and about 1 kDa, or between about 0.4 kDa and about 0.8 kDa, so that the labeling compounds do not substantially alter separation rates of the proteins in electrophoresis or chromatography, for example.
[0128]A dye used to label a selectively labeled protein of a pre-labeled protein standard set can be or comprise a chromophore, a fluorophore, or can be or comprise both a fluorophore and chromophore. The dye can comprise a chromophore that is also a fluorophore. A chromophore can be any chromophore. In some embodiments, a chromophore is a textile dye, such as for example, a Direct dye, a Disperse dye, a Dischargeable acid dye, a Kenanthol dye, a Kenamide dye, a Dyacid dye, a Kemtex reactive dye, a Kemtex acid dye, a Kemtex Easidye acid dye, a Remazol dye, a Kemazol dye, a Calcdon dye, a Cassulfon dye, an Isolan dye, a Sirius dye, an Imperon dye, a phtalogen dye, a naphtol dye, a Levafix dye, a Procion dye, and an isothiocyanate dye. Examples of textile dyes that can be used to label protein standards include, for example, Remazol brilliant blue, Uniblue A, malachite green isothiocyanate, and Orange 16 (Remazol orange).
[0129]A dye used to label a selectively labeled protein standard of a pre-labeled protein standard set can be a fluorophore. As nonlimiting examples, a fluorophore used to label a protein standard can be an Alexa fluor dye, a BODIPY dye, fluoroscein or a derivative thereof, eosin or a derivative thereof, tetramethylrhodamine, rhodamine or a derivative thereof, Texas red or a derivative thereof, pyridyloxazole or a derivative thereof, NBD chloride, NBD fluoride, ABD-F, lucifer yellow or a derivative thereof, 8-anilino-1-naphthalenesulfonic acid (8-ANS) or a derivative thereof, or Oregon green or a derivative thereof. Although some amino acids may be weakly fluorescent, they are not considered fluorophores for the purposes of the invention, in which visual detection is preferred. For purposes of the invention therefore, naturally occurring amino acids including tryptophan and tyrosine are not considered labels or labeling compounds.
[0130]Dyes can include reactive groups, such as cysteine reactive groups (e.g., maleimide, iodoacetic acid, iodoacetamide, and vinyl sulfone) or amino reactive groups (such as, for example, isothiocyanates, isocyanates, acyl azides, N-hydroxysuccinimide (NHS) esters, sulfonyl chlorides, aldehydes, ketones, glyoxals, epoxides, oxiranes, carbonaes, aryl halides, imidoesters, carbodiimides, and acid anhydrides). Reactive chemical groups such as, for example, can be added to a dye using techniques that are known in the art of organic chemistry.
[0131]A dye can be tested for suitability in labeling a protein for use as a standard by labeling a protein with the dye to be tested on a target amino acid, in which at least one non-target amino acid of the protein is depleted in the protein, and performing a separation procedure on the labeled protein and the protein in unlabeled form, detecting the labeled and unlabeled protein after the separation procedure is completed, and comparing the separation of the labeled and unlabeled protein. The method can also include staining the unlabeled protein prior to detecting the unlabeled protein. For example, the migration of a labeled protein and the unlabeled form of the same protein can be compared on an electrophoresis gel, such as an acrylamide electrophoresis gel disclosed herein, for example a 4-12%, 4-16%, or 4-20% acrylamide gradient gel, in which the molecular weight of the labeled protein whose labeled and unlabeled form are being compared is greater than about 3.5 kDa, such as at least about 5 kDa, or such as at least about 10 kDa. Migration of selectively labeled and unlabeled forms of a protein are compared under electrophoresis conditions in which the loading dye front migrates at least 5 cm from the loading site and migration of a protein calculated to be about 10 kDa and the migration of a protein calculated to about 80 kDa are at least 3 cm apart at the completion of electrophoresis. Migration of selectively labeled and unlabeled forms of a protein are preferably compared under electrophoresis conditions in which the loading dye front (for example, a Coomassie loading dye front) migrates at least 6 cm from the loading site and migration of a protein calculated to be about 10 kDa and the migration of a protein calculated to about 80 kDa are at least 3.5 cm apart at the completion of electrophoresis. Migration of selectively labeled and unlabeled forms of a protein are preferably compared under electrophoresis conditions in which a the loading dye front migrates at least 6.5 cm, for example about 6.8 cm from the loading site and migration of a protein calculated to be about 10 kDa and the migration of a protein calculated to about 80 kDa are at least 3.5 cm apart at the completion of electrophoresis.
[0132]In comparing electrophoretic migration, molecular weights of labeled and unlabeled standards are calculated based on art-recognized methods using a curve generated from plotting migration distance of proteins (or a function thereof) versus molecular weight (or a function thereof, for example, the log of molecular weight), or using point-to-point calculation based on the migration distances of two proteins of known molecular weight electrophoresed on the same gel that preferably have molecular weights that bracket the molecular weight of the analyzed protein. Electrophoretic migration of labeled and unlabeled forms of a protein standard is within a given percentage when the difference in the calculated molecular weights of the labeled and unlabeled forms of the protein using either curve-fitting of molecular weight to migration distances or point-to-point calculation are within the given percentage.
Selectively Labeled Protein Standards Depleted in Residues of a Second Amino Acid
[0133]In one aspect of the invention, a pre-labeled protein standard set includes one or more proteins selectively labeled on a first, or target, amino acid with a labeling compound, in which the one or more selectively labeled proteins is depleted in residues of a second, or non-target, amino acid that is capable of reacting with the labeling compound. A protein depleted in a non-target amino acid has fewer than one residue of a non-target amino acid per 10 kDa.
[0134]In one embodiment of this aspect, a protein of a pre-labeled protein standard set that is selectively labeled on a first amino acid comprises a naturally-occurring protein or a fragment thereof, in which the sequence of the naturally-occurring protein is depleted in residues of a non-target amino acid that is capable of reacting with the labeling compound conjugated to the target amino acid. For example, the protein that is selectively labeled can be a naturally-occurring protein that is isolated from cells, tissue, organisms, biological samples (including fluid samples, such as blood or serum), or media, where at least a portion of the protein naturally has a low abundance of a non-target amino acid. The protein can optionally be chemically or enzymatically proteolyzed to remove one or more portions of the protein, such as but not limited to a portion that includes one or more residues of a non-target amino acid. The protein that is selectively labeled can be a naturally-occurring protein that lacks a non-target amino acid and that is isolated from cells, tissue, organisms, biological samples, or media.
[0135]A selectively labeled protein depleted in a first amino acid can also be produced using recombinant methods, in which a nucleic acid sequence that encodes an amino acid sequence having homology to the sequence of a naturally-occurring protein is used to produce the protein in cells or in an in vitro synthesis system. An amino acid sequence having homology to the sequence of a naturally-occurring protein preferably has at least 70%, at least 80%, at least 90%, or at least 95% amino acid identity with at least twenty, at least thirty, at least forty, at least fifty, at least sixty, at least seventy, or at least eighty contiguous amino acids of the naturally occurring protein. In some embodiments, a selectively labeled protein has a labeling compound conjugated to a first amino acid, and includes an amino acid sequence having at least 70% homology to at least 30 contiguous amino acids of a naturally-occurring protein, in which the amino acid sequence has a reduced number of a second amino acid compared to the sequence of the naturally-occurring protein. The second amino acid is preferably a nontarget amino acid that can react with the labeling compound. In some embodiments, a selectively labeled protein is labeled on a first amino acid and includes an amino acid sequence having at least 80% homology to at least 40 contiguous amino acids of a naturally-occurring protein, in which the sequence having homology to the naturally-occurring protein has fewer residues of a second amino acid than the sequence of the naturally-occurring protein to which it is homologous. The second amino acid is preferably an amino acid that reacts with the labeling compound used to label the first amino acid.
[0136]The selectively labeled protein can, for example, be a recombinant protein that comprises one or more copies of an amino acid sequence derived from the sequence of a naturally-occurring protein that has fewer than one residue of a non-target amino acid per 10 kDa. A selectively labeled protein can include one or more copies of an amino acid sequence derived from a naturally-occurring protein that lacks a non-target amino acid.
[0137]In some embodiments, as disclosed above, the one or more selectively labeled proteins of the protein standard are made using recombinant methods, in which a protein is produced from a nucleic acid construct that comprises at least one copy of a nucleic acid sequence that encodes at least a portion of said naturally-occurring protein, in which the naturally occurring protein or portion thereof lacks residues of the second amino acid. In some embodiments, the one or more selectively labeled proteins of the protein standard are made using recombinant methods, in which a protein is produced from a nucleic acid construct that comprises at least one copy of a nucleic acid sequence that encodes at least a portion of said naturally-occurring protein, in which the nucleic acid sequence has been mutated to remove one or more codons of the second amino acid from the sequence. In some embodiments, one or more codons of the second amino acids is deleted from the nucleic acid sequence to delete amino acid residues from a standard protein that are capable of reacting with a labeling compound. In some embodiments, at least one of the one or more codons of the non-target amino acid is mutated to a codon for an amino acid other than the non-target amino acid. Mutation of a codon can be to any codon for an amino acid other than the non-target amino acid. The mutation of codons can be to any non-target codon and need not be restricted to conservative mutation. In some embodiments, mutation of a codon results in a conservative amino acid change in the amino acid sequence of the protein.
[0138]In embodiments in which the protein standard is made using recombinant methods, one or more mutations can be introduced into the nucleic acid sequence encoding the standard protein, where at least one mutation can alter a codon to change the number of residues of a target amino acid, or the position of a target amino acid. Increasing or decreasing the number of target amino acid residues can be done to optimize the number of label molecules attached to a protein standard. Codons of a target amino acid can also be mutated to optimize their position or spacing in a standard protein, which can affect labeling efficiency. Changing the position of a target amino acid in a protein can be done by altering codons and can be done to improve labeling efficiencies, for example by providing spacing between target amino acids to avoid steric hindrance during the labeling reaction, or to position a target amino acid farther from a charged group, hydrophobic region, etc. Codons of a target amino acid can be deleted, inserted, or mutated to codons of other amino acids, for example to provide proteins for labeling that include more than one target amino acid per 10 kDa, such as an average of 2, 3, 4, or more target amino acids per 10 kDa. Codons of a target amino acid can also be mutated to change the third nucleotide of the codon while retaining its amino acid specificity (through "wobble") to reduce the chance of recombination in the nucleic acid construct.
[0139]A naturally-occurring protein can be any naturally-occurring protein, and can be a prokaryotic or eukaryotic protein of any species. Proteins can be selected based on properties such as abundance in cells in which they are produced, ease of isolation, or sequence properties, such as, but not limited to, the abundance or accessibility of residues a target amino targeted for labeling in the sequence, or the lack of abundance of additional non-target amino acid(s) in the sequence. All or one or more portions of a sequence of a naturally-occurring protein can be used in a protein standard, or can be selected as a protein whose sequence can be mutated for engineering a protein for use as a selectively labeled protein standard.
[0140]For example, in some preferred embodiments of a pre-labeled protein standard, the target amino acid is lysine, and a non-target amino acid is cysteine. In this case protein sequences can optionally be selected base on the abundance of lysine and the paucity of cysteine in the amino acid sequence used, which in some embodiments can reduce the number of codons to be mutated. The amino acid sequence encoding the protein sequence can optionally be mutated to further reduce the number of residues of cysteine and/or other non-target amino acids, for example, histidine and/or tryptophan, which can be labeled in reactions that target lysine.
[0141]In other embodiments of a pre-labeled protein standard, the target amino acid is cysteine and a second amino acid is lysine. In this case protein sequences can optionally be selected base on the abundance of cysteine and the paucity of lysine in the amino acid sequence used, which in some embodiments can reduce the number of codons to be mutated.
[0142]In alternative embodiments, a selectively labeled protein that is depleted in a non-target amino acid can in some embodiments be a protein that comprises an amino acid sequence that has no known homology to a naturally-occurring protein, and can be designed and synthesized recombinantly or chemically, or using a combination of chemistry and recombinant technologies. A selectively labeled protein that is comprises sequence not derived from a naturally-occurring protein can in some preferred embodiments lack residues of a non-target amino acid.
[0143]Protein sequences lacking one non-target amino acid can also be further selected based on a low frequency of other potential non-target amino acids. For example, where lysine is a target amino acid to be conjugated with a dye, histidine and tryptophan, which are less reactive than lysine and cysteine but nonetheless can react with amino-reactive groups of labeling compounds, can optionally be considered non-target amino acids in addition to cysteine. In the present example, sequences lacking cysteine can optionally be analyzed for the frequency of these amino acids in the sequence as well.
[0144]A pre-labeled protein standard set can comprise a selectively labeled protein that comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more copies of an amino acid sequence that is depleted in a non-target amino acid. In illustrative embodiments, the sequence lacks residues of a non-target amino acid. The amino acid sequence depleted or deficient in a non-target amino acid can be a designed sequence that lacks homology to a known naturally-occurring protein, or can be a sequence having homology to an amino acid sequence of a naturally-occurring protein, for example, having at least 70% homology to at least 30 contiguous amino acids of a naturally occurring protein, at least 80% homology to at least 40 contiguous amino acids of a naturally occurring protein, at least 80% homology to at least 50 contiguous amino acids of a naturally occurring protein. In some preferred embodiments of the invention, a pre-labeled protein standard set can include two or more selectively labeled proteins, in which the two or more selectively labeled proteins include a labeling compound conjugated to a first amino acid, and comprise different numbers of copies of an amino acid sequence that is depleted in or deficient in a second amino acid. The second amino acid is preferably a non-target amino acid that reacts with a labeling compound used to label the selectively labeled protein.
[0145]In one aspect, the invention includes a pre-labeled protein standard set that includes two or more proteins selectively labeled on a first amino acid with a labeling compound and depleted in a second amino acid capable of reacting with the labeling compound, in which the two or more selectively labeled proteins includes different numbers of copies of an amino acid sequence having at least 70% homology to at least 30 contiguous amino acids of a sequence of a naturally-occurring protein. The pre-labeled protein standard set can include two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more selectively labeled proteins that comprises different numbers of copies of an amino acid sequence that is depleted in residues of a second amino acid.
[0146]In one embodiment, a pre-labeled protein standard set of the invention comprises two or more proteins of different molecular weights that are labeled on lysine and depleted in cysteine residues. The invention includes in some illustrative embodiments a set of pre-labeled protein standards that includes at least two proteins of different molecular weight that are labeled on lysine and lack cysteine residues. The proteins selectively labeled on lysine can be isolated from cells, tissue, organisms, biological samples, or media, or can be made using recombinant methods. Using recombinant methods, proteins can be synthesized for use as selectively labeled standards, in which the proteins comprise one or more copies of a sequence that is depleted in or lacks cysteine. For example, a selectively labeled protein can comprise one or more copies of a sequence from the C-terminus of one or more ADP-ribosylation factors (Schurmann et al. Journal of Biological Chemistry 269: 15683 (1994)) or a sequence of one or more Bacillus megaterium spore proteins that lack cysteine residues (Setlow, Journal of Biological Chemistry 250: 8168 (1975)). Such sequences can be fused in any combination with themselves or other sequences to provide protein standards. Other amino acid sequences that lack cysteine can be found by searching gene or protein databases. Sequences lacking cysteine can be further selected based on the frequency residues of the target amino acid (e.g., lysine).
[0147]In some embodiments, the invention provides pre-labeled protein standard sets having a plurality of proteins selectively labeled on lysine and lacking cysteine, in which two or more selectively labeled proteins comprise one or more copies of an amino acid sequence that is depleted in cysteine. The pre-labeled protein standard set can include two or more, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more proteins that are selectively labeled on lysine and lack depleted in cysteine, in which the selectively labeled proteins comprise one or more copies of an amino acid sequence depleted in cysteine. A protein standard selectively labeled on lysine is labeled with a labeling compound that comprises an amino-reactive group, such as, but not limited to, an isothiocyanate, an isocyanate, an acyl azide, an N-hydroxysuccinimide (NHS) ester, a sulfonyl chloride, an aldehyde, a ketone, a glyoxal, an epoxide, an oxirane, a carbonate, an aryl halide, an imidoester, a carbodiimides, or an acid anhydrides. A protein standard selectively labeled on lysine is preferably labeled with a dye that comprises an amino-reactive group.
[0148]In another embodiment, a pre-labeled protein standard set of the invention comprises two or more proteins of different molecular weights that are labeled on cysteine and depleted in lysine residues. The invention includes in some illustrative embodiments a set of pre-labeled protein standards that includes at least two proteins of different molecular weight that are labeled on cysteine and lack lysine residues. A selectively labeled protein can be a naturally-occurring protein isolated from cells, tissue, organisms, biological samples, or media, or can be made using recombinant methods. For example, using recombinant methods, sequences of proteins having at least a portion of the protein having fewer than one lysine per 10 kDa of protein, such as, for example, sequences encoding seed storage proteins of cereal crops (such as, for example, the zein proteins of maize, the gliadins of wheat), the L domain of HIV or Ebola viruses, or the WNK-1 and WNK-4 proteins (Coleman et al. Proc. Natl. Acad. Sci. 94: 709994-97 (1997); Shimoni et al. Journal of Biological Chemistry 271: 18869-18874 (1996); Yang et al J. Clin. Invest. 115: 1379-1387 (2005)) can be fused in any combination to provide protein standards. Other amino acid sequences that lack or are depleted in lysine can be found by searching gene or protein databases. Sequences depleted in a non-target amino acid can be further selected based on the frequency of the target amino acid, e.g., cysteine. Sequences depleted in lysine can be further selected based on low frequency of other potential non-target amino acids, such as, but not limited to, histidine or tryptophan.
[0149]In other embodiments, the invention provides pre-labeled protein standard sets having a plurality of proteins selectively labeled on cysteine and lacking lysine, in which two or more selectively labeled proteins comprise one or more copies of an amino acid sequence depleted in lysine. The pre-labeled protein standard set can include two or more, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more proteins that are selectively labeled on cysteine and are depleted in lysine, in which the selectively labeled proteins comprise one or more copies of an amino acid sequence depleted in lysine. A protein standard selectively labeled on cysteine is labeled with a labeling compound that comprises an sulfhydryl-reactive group, such as, but not limited to, vinyl sulfone, iodoacetamide, maleimide, or iodoacetic acid. A protein standard selectively labeled on lysine is preferably labeled with a dye that comprises an sulfhydryl-reactive group.
Selectively Labeled Protein Standards Comprising an Amino Acid Sequence Derived from a Naturally-Occurring Protein
[0150]In one aspect, the invention includes pre-labeled protein standard sets that have one or more selectively labeled proteins, in which a selectively labeled protein comprises a labeling compound conjugated to a first amino acid, and comprises one or more copies of an amino acid sequence derived from a naturally-occurring protein, in which the amino acid sequence derived from a naturally-occurring protein has a reduced number of residues of a second amino acid capable of interacting with the labeling compound relative to the wild-type amino acid sequence of the naturally occurring protein.
[0151]An amino acid sequence derived from the sequence of a naturally-occurring protein preferably has at least 70%, at least 80%, at least 90%, or at least 95% amino acid identity with at least twenty, at least thirty, at least forty, at least fifty, at least sixty, at least seventy, or at least eighty contiguous amino acids of the naturally occurring protein.
[0152]In certain exemplary embodiments, a protein selectively labeled on a first amino acid is a recombinant protein made from a nucleic acid construct, and one or more codons for one or more non-target amino acids is mutated or deleted from the nucleic acid sequence of the construct encoding the amino acid sequence with homology to an amino acid sequence of a naturally-occurring protein. For example, an engineered protein to be used for making pre-labeled protein standards can have one or more copies of an amino acid sequence with at least 70% or at least 80% identity with at least 20, at least 30, at least 40, or at least 50 contiguous amino acids of a thioredoxin sequence, in which lysine has been removed from the sequence by deletion or mutation of lysine codons in the nucleic acid sequence encoding the protein. Lysine codons can be mutated to any nonlysine codons. In some instances, one or more lysine codons is mutated to a nonlysine codon based on the hydrophilicity, charge, or reactivity of the nonlysine amino acid to optimize properties such as solubility or purification of the labeled protein.
[0153]The invention provides molecular weight standard sets in which two or more selectively labeled proteins of different molecular weights comprise different numbers of copies of an amino acid sequence having homology to an amino acid sequence of a naturally-occurring protein. For example, a standard set can have proteins selectively labeled on a target amino acid having two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more copies of an amino acid sequence that is at least 70% or at least 80% identical to at least 20, at least 30, at least 40, or at least 50 contiguous amino acids of a naturally-occurring protein which lack residues of a non-target amino acid that are present in the wild-type protein sequence.
[0154]A naturally-occurring protein can be any naturally-occurring protein. Nucleotide-disulfide oxidoreductases are highly soluble proteins (an advantage for accessibility of residues for labeling) having an abundance of cysteine residues. Examples of nucleotide-disulfide oxidoreductases include lipoamide dehydrogenase, glutathione reductase, or thioredoxin. All or a portion of the amino acid sequence of a lipoamide dehydrogenase, glutathione reductase, or thioredoxin can be incorporated into a protein for use as a pre-labeled protein standard that is selectively labeled on cysteine. A lipoamide dehydrogenase, glutathione reductase, and/or thioredoxin whose sequence is used for engineering a pre-labeled protein standard can be from a prokaryotic or eukaryotic source. An nucleotide-disulfide oxidoreductases can be, as nonlimiting examples, any of SEQ ID NO:1 (E. coli thioredoxin), SEQ ID NO:2 (human thioredoxin), SEQ ID NO:3 (E. coli glutaredoxin 1), SEQ ID NO:3 (E. coli glutaredoxin 2), SEQ ID NO:5 (E. coli glutathione oxidoreductase), SEQ ID NO:6 (human glutathione oxidoreductase), SEQ ID NO:7 (E. coli lipoamide dehydrogenase), SEQ ID NO:8 (human lipoamide dehydrogenase), their variants, their analogues in other species, and variants of such analogues.
[0155]In some preferred embodiments of the invention, a protein used as a pre-labeled molecular weight standard includes one or more copies of an amino acid sequence derived from a lipoamide dehydrogenase, glutathione reductase, and/or thioredoxin sequence. In some preferred embodiments of the invention, a protein used as a pre-labeled molecular weight standard includes one or more copies of an amino acid sequence derived from a thioredoxin sequence. In some preferred embodiments of the invention, a protein used as a pre-labeled molecular weight standard includes one or more copies of an amino acid sequence derived from a bacterial thioredoxin sequence, such as an E. coli thioredoxin sequence, and can be a low molecular weight thioredoxin, such as a sequence encoded by TrxA.
[0156]All or a portion of a thioredoxin sequence can be used in making one or more pre-labeled protein standards. For example, a thioredoxin sequence used in a protein standard can have a truncation of from one to 50 amino acids from the carboxy terminus, such as, for example, from one to ten, from ten to twenty, form twenty to thirty, form thirty or forty, or from forty to fifty, amino acids can be truncated from the carboxy terminus. In some preferred embodiments, 22 amino acids are truncated from the end of a thioredoxin sequence, such as a bacterial thioredoxin sequence used as a sequence in a protein standard. In some preferred embodiments, from 39-41 amino acids are truncated from the end of a thioredoxin sequence, such as a bacterial thioredoxin sequence used as a sequence in a protein standard.
[0157]In some embodiments, a protein of a pre-labeled protein standard set that is selectively labeled on cysteine comprises an amino acid sequence derived from an nucleotide-disulfide oxidoreductase, such as a lipoamide dehydrogenase, a glutathione reductase, or a thioredoxin. In some preferred embodiments, an amino acid sequence is derived from a thioredoxin sequence, having at least 70% or at least 80% identity with the amino acid sequence of at least 20, at least 30, at least 40 or at least 50 amino acids of a thioredoxin, such as a truncated thioredoxin. In some preferred embodiments, an amino acid sequence derived from a thioredoxin sequence differs from the naturally-occurring thioredoxin sequence by lacking lysine residues. In some preferred embodiments, a selectively labeled pre-labeled protein standard is devoid of lysine residues and is labeled on one or more cysteine residues, and comprises one or more copies of an amino acid sequence derived from a thioredoxin. In preferred embodiments, the protein is made from a nucleic acid construct that includes a nucleic acid sequence encoding one or more copies of an amino acid sequence derived from a naturally-occurring thioredoxin sequence, in which the nucleic acid sequence has been mutated to delete one or more lysine codons or to change one or more lysine codons to non-lysine codons.
[0158]In some aspects of the invention, a pre-labeled protein standard set can include one or more copies of an amino acid sequence having at least 70% or at least 80% identity to at least 20, at least 30, at least 40, or at least 50 contiguous amino acids of a naturally-occurring protein in which the amino acid sequence comprises one or more amino acid changes that alter the number or spacing of a first amino acid targeted for labeling.
[0159]Additional target amino acid codons can be added to a nucleic acid sequence that encodes a protein standard of the invention. In a preferred embodiment, one or more additional cysteine codons is added to a nucleic acid sequence encoding a truncated thioredoxin. Two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more copies of the nucleic acid sequence encoding a truncated thioredoxin can be assembled together to make a recombinant protein having multiple copies of a truncated thioredoxin sequence. In some embodiments, the recombinant nucleic acid constructs used to produce the protein standards are further mutated to allow alternate codon usage for the same amino acid from copy to copy to reduce the risk of genetic recombination.
Pre-Labeled Proteins Having Consistent Ratios of a First Amino Acid to Molecular Weight
[0160]In some preferred embodiments of the invention, a pre-labeled protein standard set includes two or more proteins of different molecular weights labeled on a target amino acid, in which the ratios of the number of residues of the target amino acid to molecular weight of two or more of the selectively labeled proteins are within 5% of one another, in some embodiments within 2.5% of one another. For example, the ratio of the number of residues of a target amino acid to molecular weight may be 4 residues per 10 kDa, or 0.4 residues of first amino acid/kDa for a first protein of a standard set, and can be, for example, between 0.38 and 0.42 residues of target amino acid/kDa for a second protein of a standard set, where the first and second proteins have ratios of the number of target amino acid residues to molecular weight that are within 5% of one another.
[0161]In some preferred embodiments, the two or more labeled proteins that have a consistent ratio of the number of residues of a first, or target, amino acid to molecular weight of the proteins are selectively labeled on a first amino acid. In some preferred embodiments, the two or more labeled proteins are selectively labeled on a first amino acid and comprise one or more copies of an amino acid sequence of a naturally-occurring protein or having at least 70% or at least 80% identical to at least 20, at least 30, at least 40, or at least 50 contiguous amino acids of a naturally-occurring protein. In some preferred embodiments, the two or more labeled proteins are comprise a labeling compound bound to a first amino acid and comprise one or more copies of an amino acid sequence of or having homology to an amino acid sequence of a naturally-occurring protein, in which the amino acid sequences of the labeled proteins lacks residues of a second amino acid that can react with the labeling compound. The invention provides pre-labeled protein standard sets that comprise a plurality of labeled proteins, in which two or more of the labeled proteins are selectively labeled on a first amino acid with a labeling compound and lack residues of a second amino acid that is capable of reacting with the labeling compound, in which the ratios of the number of residues of the first amino acid to molecular weight of the two or more selectively labeled proteins are within 5%, 2.5%, or 1% of one another.
[0162]In some aspects of a pre-labeled protein standard set, the set comprises a plurality of labeled proteins, and at least two proteins of the set are labeled on a target amino acid and have an average of between one and ten residues of the target amino acid per 10 kDa, such as an average of between two and seven residues of the target amino acid, such as an average of between three and five residues of the target amino acid, such as an average of between 3.5 and 4.5 residues of the target amino acid per 10 kDa. Preferably, in these embodiments, the two or more proteins labeled on a target amino acid are selectively labeled with a labeling compound on the target amino acid. In exemplary embodiments, the selectively labeled protein lacks residues of a non-target amino acid capable of reacting with the dye. In some preferred embodiments, a pre-labeled protein standard set comprises at least five labeled proteins, in which three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more of the proteins are selectively labeled on a target (first) amino acid, and have an average of between one and ten residues of the target amino acid per 10 kDa, such as an average of between two and seven residues of the target amino acid, such as an average of between three and five residues of the target amino acid, such as an average of between 3.5 and 4.5 residues of the target amino acid per 10 kDa.
[0163]In some preferred embodiments, a pre-labeled protein standard set comprises at least five labeled proteins, in which three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more of the proteins are labeled on cysteine, and have an average of between three and five cysteine residues, such as an average of between 3.5 and 4.5 cysteine residues per 10 kDa. In some preferred embodiments, a pre-labeled protein standard set comprises at least five labeled proteins, in which three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more of the proteins lack lysine and are labeled on cysteine, and have an average of between three and five residues of cysteine, such as between 3.5 and 4.5 residues of cysteine, per 10 kDa.
[0164]Proteins of a pre-labeled protein standard set that are labeled with a labeling compound on a target amino acid and have ratios of the number of residues of the target amino acid to molecular weight that are within 5% of one another can have molecular weights that differ from one another by at least 10 kDa, at least 20 kDa, at least 30 kDa, at least 40 kDa, at least 50 kDa, at least 60 kDa, at least 70 kDa, at least 80 kDa, at least 90 kDa, at least 100 kDa, at least 110 kDa, or at least 150 kDa, where the given molecular weights are plus or minus 1 kDa. Proteins of a pre-labeled protein standard set that are labeled with a dye on a target amino acid and have ratios of the number of residues of the target amino acid to molecular weight that are within 5% of one another can be labeled with the same dye, or with different dyes.
[0165]The invention provides in a further aspect a pre-labeled protein standard set that comprise a plurality of labeled proteins span a molecular weight range of from 10 kDa or less to 100 kDa or greater, in which two, three, four, five or more of the plurality of labeled proteins are selectively labeled with a dye on a first amino acid and have ratios of a first amino acid to molecular weight that are within 5% of one another, in which the migration of the five or more pre-labeled protein standards in acrylamide gel electrophoresis under denaturing conditions does not differ substantially from the migration of the same set of proteins in unlabeled form. In preferred embodiments, each of the five or more labeled protein standards that has a molecular weight of 10 kDa or greater migrates within 5% of each of the five or more proteins in unlabeled form on the same acrylamide gels.
[0166]A pre-labeled protein standard set of the invention can include two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more labeled proteins. For example, pre-labeled protein standard sets can have between ten and fifteen, between fifteen and twenty, twenty or more, thirty or more, forty or more, fifty or more sixty or more, seventy or more, eighty or more, ninety or more, or one hundred or more labeled proteins. All or a subset of the labeled proteins of a pre-labeled protein standard set can be selectively labeled. Two or more of the labeled proteins of a pre-labeled protein standard set can comprise a labeling compound on a target amino acid and have ratios of the number of residues of the target amino acid to molecular weight that are within 5% of one another. A pre-labeled protein standard set of the invention can include two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or more proteins selectively labeled on a target amino acid.
[0167]In some preferred embodiments, a pre-labeled standard set comprises a plurality of labeled proteins, in which at least two of the proteins are selectively labeled on a target amino acid, and the at least two proteins selectively labeled on a target amino acid have ratios of the number of target amino acid residues to molecular weight that are within 5% of one another.
[0168]As a nonlimiting example, a pre-labeled protein standard set can comprise from five to twenty labeled proteins, of which from one to twenty are labeled on cysteine and lack lysine residues. As a nonlimiting example, a pre-labeled protein standard set can comprise from five to twenty labeled proteins, of which from two to twenty comprise a label on cysteine residues and lack lysine residues, and have ratios of cysteine residue number to molecular weight that are within 5% of one another.
Highly Resolving Electrophoretic Separation of Pre-Labeled Protein Standards
[0169]Preventing the reaction of a labeling compound with a non-target amino acid can reduce the inconsistency in labeling of a protein. For example, labeling of a particular protein with a dye that has high specificity for a first amino acid and reduced specificity for a second amino acid can result in a population of labeled protein variants, in which the variants are predominantly labeled on the first amino acid, but vary in the degree of labeling of the second amino acid that is present on the protein. Such variability in the population of labeled protein results in a range of masses for the particular labeled protein, depending on the range in the amount of dye molecules attached to the protein. This leads to a protein standard having variable label intensity per microgram of protein, and poor resolution of the protein standard in separation techniques that rely on mass, such as, but not limited to, electrophoresis and chromatography.
[0170]The present invention provides protein standards that are pre-labeled that separate based on size, charge, or a combination of size and charge, distinctly and consistently. Pre-labeled standards are labeled prior to separation or experimental procedures, and can be observed during or after separation procedures without performing additional steps required to stain the proteins in the midst of or at the conclusion of a separation or experimental procedure. Pre-labeled protein standards can be used in protein separation techniques such as, but not limited to, isolelectric focusing in semi-solid (e.g. gel) or liquid media; chromatography, including chromatographic separation based on size, charge, or a combination thereof, including HPLC and FPLC; and electrophoresis, including, without limitation, capillary electrophoresis, free-flow electrophoresis, non-denaturing (native) gel electrophoresis and denaturing gel electrophoresis, mass spectrometry, and chromatofocusing. In some preferred aspects, the present invention provides protein molecular weight standards that are selectively labeled, such that attachment of a dye to an amino acid that is not targeted for labeling (a non-target amino acid) is restricted. The invention additionally provides sets of pre-labeled protein standards that can be used as molecular weight markers in biochemical separations, in which at least one labeled protein of the sets is selectively labeled on a first amino acid.
[0171]The pre-labeled protein standards of the present invention are particularly useful in gel electrophoresis, in which molecular weights can be determined using the pre-labeled standards run alongside one or more sample proteins. For example, pre-labeled standards provided herein can be used as markers in Blue Native gel electrophoresis, in which non-denatured proteins are separated based on size (described in Schagger H and von Jagow G (1991) Anal. Biochem. 199: 223-231; Schagger H, Cramer W A, and von Jagow G (1994) Anal. Biochem. 217: 220-230; and Schagger H (2001) Methods Cell Biol. 65: 231-244), or can be used in denaturing gel electrophoresis, such as denaturing polyacrylamide gel electrophoresis in which proteins are denatured using urea, formamide, or one or more denaturing detergents, such as, but not limited to, sodium dodecyl sulfate (SDS) or lithium dodecyl sulfate (LDS). In some preferred embodiments, proteins standards are used in denaturing acrylamide gel electrophoresis in which proteins are denatured using a detergent, such as but not limited to SDS or LDS. Many denaturing polyacrylamide gel electrophoresis systems are known in the art, such as, for example, Bis-Tris gels, Tris-tricine gels, Tris-acetate gels, or Tris-glycine gels. Gels for electrophoretic separation of proteins are available commercially, for example, NuPAGE® Novex® Tris-Acetate gels, NuPAGE® Novex® Bis-Tris gels, Novex® Tricine gels, and Novex® Tris-Glycine gels, all available from Invitrogen Corp., Carlsbad, Calif. The invention provides pre-labeled protein standards that can be used as molecular weight markers, in which the pre-labeled protein standards produce sharp bands on electrophoresis gels, such as electrophoresis gels run under denaturing conditions, and the migration of the pre-labeled protein standards are substantially the same as the migration of their unlabeled counterparts.
[0172]Migration of labeled and unlabeled forms of a protein can be compared, for example, on Bis-Tris acrylamide gels using MOPS or MES buffer, or on Tris-acetate, Tricine, or Tris-glycine acrylamide gels, under electrophoresis conditions in which a the loading dye front migrates at least 5 cm from the loading site and migration of a protein calculated to be about 10 kDa and the migration of a protein calculated to be about 80 kDa are at least 3.5 cm apart at the completion of electrophoresis. The gels can be "mini gels" having lengths of 10 cm or less, such as, for example, gels 8 cm in length, or can be more than 10 cm in length, for example 12 cm, 15, cm, 20 cm or greater in length, in which the dye front at the end of the electrophoresis period has migrated at least 80% the length of the gel. The dye front can be a Coomassie dye front, such as a Coomassie G250 dye front.
[0173]For example, to test the consistency of migration between a labeled protein standard and its unlabeled counterpart, electrophoresis can be performed on a polyacrylamide gel, having a length of 8 cm, in which at the end of electrophoresis the dye front of the gel has migrated at least 5 cm, such as at least 6 cm, such as at least 6.5 cm, such as about 6.8 cm, from the loading site. The dye front can be a Coomassie dye front, such as a Coomassie G250 dye front.
[0174]For example, 4-12% NuPAGE® Bis-Tris acrylamide 8 cm×8 cm gels using MOPS or MES buffer, or 4-20% Tris-glycine 8 cm×8 cm acrylamide gels available from Invitrogen (Carlsbad, Calif.) can be used to determine migration properties of labeled and unlabeled protein standards using electrophoresis conditions provided in the manufacturer's manual for separating proteins. Migration of selectively labeled and unlabeled forms of a protein are compared under electrophoresis conditions in which a the loading dye front migrates at least 5 cm from the loading site and migration of a protein calculated to be about 10 kDa and the migration of a protein calculated to about 80 kDa are at least 3 cm apart at the completion of electrophoresis. Migration of selectively labeled and unlabeled forms of a protein are preferably compared under electrophoresis conditions in which a the loading dye front migrates at least 6 cm from the loading site and migration of a protein calculated to be about 10 kDa and the migration of a protein calculated to about 80 kDa are at least 3.5 cm apart at the completion of electrophoresis. Migration of selectively labeled and unlabeled forms of a protein are preferably compared under electrophoresis conditions in which a the loading dye front migrates at least 6.5 cm from the loading site and migration of a protein calculated to be about 10 kDa and the migration of a protein calculated to about 80 kDa are at least 3.5 cm apart at the completion of electrophoresis. The dye front can be a Coomassie dye front, such as a Coomassie G250 dye front.
[0175]Preferably, the calculated molecular weights for a pre-labeled protein standard having a molecular weight greater than 5 kDa and its unlabeled counterpart on one of the referenced denaturing acrylamide gels are within 10%, 7%, or 5% of one another.
[0176]The invention provides pre-labeled protein standard sets having five or more labeled proteins of different molecular weights, in which all of the pre-labeled proteins having a molecular weight of greater than 3.5 kDa (such as, for example, having a molecular weight of greater than 5 kDa, such as, for example, having a molecular weight of 10 kDa or greater) have substantially the same migration on electrophoresis gels as their unlabeled counterparts.
[0177]The invention provides sets of pre-labeled protein standards having at least ten, at least eleven, at least twelve, or at least fifteen pre-labeled proteins of different molecular weights, in which all of the pre-labeled proteins of the sets having a molecular weight of greater than 3.5 kDa, greater than 5 kDa, or 10 kDa or greater, migrate on electrophoresis gels, such as for example Bis-Tris gels and Tris-glycine gels as they are known in the art, within 10%, 7%, or 5% of the migration unlabeled counterparts.
[0178]The invention provides individual pre-labeled proteins that migrate within 10%, within 7%, within 5%, within 4%, within 2.5%, within 2%, within 1.5%, or within 1% of the migration distance of the same proteins that are not labeled.
[0179]The invention provides pre-labeled protein molecular weight standard sets in which all the proteins of the set having a molecular weight of greater than or equal to 3.5 kDa migrate within 5% of the migration distance of the same proteins that are not labeled. The invention provides protein molecular weight standard sets in which all the proteins of the set having a molecular weight of greater than or equal to 5 kDa migrate within 5% of the migration distance of the same proteins that are not labeled. The invention provides protein molecular weight standard sets in which all the proteins of the set having a molecular weight of 10 kDa or greater migrate within 5% of the migration distance of the same proteins that are not labeled.
[0180]The invention provides pre-labeled protein molecular weight standard sets in which all the proteins of the set having a molecular weight of greater than or equal to 3.5 kDa migrate within 4%, within 2.5%, within 2%, within 1.5%, or within 1% of the migration distance of the same proteins that are not labeled under standard protein gel electrophoresis conditions on a 4-12% Bis-Tris gel or a 4-20% Tris-glycine gel. The invention provides pre-labeled protein molecular weight standard sets in which all the proteins of the set having a molecular weight of greater than or equal to 5 kDa migrate within 4%, within 2.5%, within 2%, within 1.5%, or within 1% of the migration distance of the same proteins that are not labeled. The invention provides pre-labeled protein molecular weight standard sets in which all the proteins of the set having a molecular weight of 10 kDa or greater migrate within 4%, within 2.5%, within 2%, within 1.5%, or within 1% of the migration distance of the same proteins that are not labeled.
[0181]The invention further provides pre-labeled protein molecular weight standard sets in which all the proteins of the set having a molecular weight of greater than 3.5 kDa, greater than 5 kDa, or greater than or equal to 10 kDa, migrate within 4%, within 2.5%, within 2%, within 1.5%, or within 1% of the migration distance of the same proteins that are not labeled.
[0182]In some aspects of the invention, a pre-labeled protein standard set comprises from two to twenty proteins, in which two or more of the proteins are selectively labeled, such that when the a pre-labeled protein standard set is electrophoresed on a denaturing acrylamide gel, such as an 8 cm long Bis-Tris gel run with MES electrophoresis buffer (for example, a 4-12% Bis-Tris 8×8 cm gel, Invitrogen, Carlsbad, Calif.), the widths of bands from proteins having a molecular weight of greater than or equal to 10 kDa differ by less than 2-fold. In some embodiments, pre-labeled protein standard set comprises labeled proteins ranging in size from 10 kDa or less to 100 kDa or more, and the width of visible bands visible to the naked eye from proteins having a molecular weight of at least 10 kDa to 100 kDa or more differ in width by less than 50%, less than 40%, or less than 30%. The width of bands visible to the naked eye from proteins having a molecular weight of at least 20 kDa to less than 100 kDa can differ in width by 15% or less. A pre-labeled standard set of the invention can include at least 6 proteins comprising at least four different dyes having different colors having a molecular weight of at least 20 kDa to less than 100 kDa, in which the width of the bands visible to the naked eye of the electrophoresed proteins differ by less than 15%. A pre-labeled standard set include 5 proteins in which the width of bands visible to the naked eye of the electrophoresed proteins difference by 3% or less. A pre-labeled standard set include 5 proteins labeled with at least four different dyes of different colors, in which the width of bands visible to the naked eye of the electrophoresed proteins difference by 3% or less.
[0183]In one embodiment, a pre-labeled protein standard set includes 6 proteins stained with four different dyes having distinguishably different colors, in which the proteins have a molecular weight of at least 20 kDa to less than 100 kDa, in which the width of bands of the electrophoresed proteins difference by less than 15%. In another embodiment, a pre-labeled protein standard set includes 5 proteins stained with four different dyes having distinguishably different colors, in which the proteins have a molecular weight of from about 20 kDa to about 80 kDa, in which the molecular weights differ of the 5 proteins differ by equal increments, in which the width of bands of the electrophoresed proteins differ by 3% or less.
Pre-Labeled Protein Standard Sets
[0184]The invention provides in a further aspect a pre-labeled protein standard set that comprises five or more labeled protein standards that span a molecular weight range of from 10 kDa or less to 100 kDa or greater, in which the migration of the five or more labeled protein standards in denaturing acrylamide gel electrophoresis does not differ substantially from the migration of the same set of proteins in unlabeled form. In preferred embodiments, the electrophoretic migration of each of the five or more labeled protein standards that have a molecular weight of 10 kDa or greater is within 5% of the electrophoretic migration of each of the five or more labeled protein standards calculated from the same acrylamide gels. In some embodiments, a pre-labeled protein standard set includes five, six, seven, eight, nine, ten, eleven, twelve or more labeled proteins, in which the labeled proteins span a molecular weight range of from 10 kDa or less to at least 100 kDa, in which electrophoretic migration on acrylamide gels of each of the five or more labeled protein standards having a molecular weight of 10 kDa or greater is within 5% of the electrophoretic migration of each of the five or more protein standards in unlabeled form on the same acrylamide gels.
[0185]In some embodiments, a pre-labeled protein standard set includes at least ten labeled proteins spanning a molecular weight range of from 10 kDa or less to 250 kDa or greater, in which the electrophoretic migration of each of the labeled protein standards having a molecular weight of 10 kDa or greater is within 5% of the electrophoretic migration of each of the protein standards in unlabeled form. In some embodiments, a pre-labeled protein standard set includes at least ten labeled proteins spanning a molecular weight range of from 10 kDa or less to 250 kDa or greater, in which the electrophoretic migration of 90% of the labeled protein standards having a molecular weight of 10 kDa or greater is within 4% of the electrophoretic migration of each of the protein standards in unlabeled form. In some embodiments, a pre-labeled protein standard set includes at least ten labeled proteins spanning a molecular weight range of from 10 kDa or less to 250 kDa or greater, in which the electrophoretic migration of 70% of the labeled protein standards having a molecular weight of 10 kDa or greater is within 3% of the electrophoretic migration of each of the protein standards in unlabeled form. In some embodiments, a pre-labeled protein standard set includes at least ten labeled proteins spanning a molecular weight range of from 10 kDa or less to 250 kDa or greater, in which the electrophoretic migration of 70% of the labeled protein standards having a molecular weight of 10 kDa or greater is within 2.5% of the electrophoretic migration of each of the protein standards in unlabeled form. In some embodiments, a pre-labeled protein standard set includes at least ten labeled proteins spanning a molecular weight range of from 10 kDa or less to 250 kDa or greater, in which the electrophoretic migration of 70% of the labeled protein standards having a molecular weight of 10 kDa or greater is within 2% of the electrophoretic migration of each of the protein standards in unlabeled form.
[0186]The pre-labeled protein molecular weight standard sets can comprise two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or more labeled proteins. For example, pre-labeled protein standard sets can have between ten and fifteen, between fifteen and twenty, twenty or more, thirty or more, forty or more, fifty or more sixty or more, seventy or more, eighty or more, ninety or more, or one hundred or more labeled proteins. Any or all of the of the proteins of a pre-labeled protein molecular weight standard set can be selectively labeled. For example, a pre-labeled protein molecular weight standard sets can comprise two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or more labeled proteins, of which one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or more are selectively labeled on a target amino acid. A pre-labeled protein molecular weight standard sets can comprise between ten and fifteen, between fifteen and twenty, twenty or more, thirty or more, forty or more, fifty or more sixty or more, seventy or more, eighty or more, ninety or more, or one hundred or more labeled proteins, of which between ten and fifteen, between fifteen and twenty, twenty or more, thirty or more, forty or more, fifty or more sixty or more, seventy or more, eighty or more, ninety or more, or one hundred or more are selectively labeled on a target amino acid.
[0187]In some embodiments of these aspects, one, two, three, four, five, or more than five labeled proteins of a protein standard set having molecular weights of 10 kDa or more are selectively labeled on a target amino acid and migrate substantially the same as their unlabeled counterparts. In some embodiments, one, two, three, four, five, or more than five labeled proteins of the protein standard set are selectively labeled on lysine and lack cysteine residues. In some embodiments of this aspect, one, two, three, four, five, or more than five labeled proteins of the protein standard set are selectively labeled on cysteine and lack lysine residues.
[0188]In other examples, a pre-labeled protein standard set can comprise from two to twenty labeled proteins, of which from one to twenty are labeled on lysine and lack cysteine residues, and, optionally, additionally lack one or more of one or more histidine residues, one or more tryptophan residues, or one or more tyrosine residues. In another example, a pre-labeled protein standard set can comprise from five to twenty labeled proteins, of which from two to twenty are labeled on lysine and lack cysteine residues, and, optionally, additionally lack one or more of one or more histidine residues, tryptophan residues, or one or more tyrosine residues, and have ratios of lysine residue number to molecular weight that are within 5% of one another.
[0189]Different proteins of a pre-labeled protein standard set can be labeled on different amino acids. Different proteins of a pre-labeled protein standard set can be labeled with different dyes having different colors, such that two or more protein bands can be distinguished by color when the proteins of the standard set are separated, such as on a gel. For example, two, three, four, or more different dyes can be used, such that one or more of the labeled proteins are labeled with a first dye and one or more of the labeled proteins are labeled with a second dye; or such that one or more of the labeled proteins are labeled with a first dye, one or more of the labeled proteins are labeled with a second dye, and one or more of the labeled proteins are labeled with a third dye; or such that one or more of the labeled proteins are labeled with a first dye, one or more of the labeled proteins are labeled with a second dye, one or more of the labeled proteins are labeled with a third dye, and one or more of the labeled proteins are labeled with a fourth dye, etc. Where multiple dyes are used to label proteins of a pre-labeled protein standard set, one, two, three, four, or more pre-labeled proteins of the set can be labeled with the same dye.
[0190]In some embodiments, pre-labeled protein standard set of the invention can span any molecular weight range, but in preferred embodiments spans a molecular weight range of from 10 kDa or less to 100 kDa or greater, or from 10 kDa or less to 150 kDa or greater, or from 5 kDa or less to 150 kDa or greater, or from 10 kDa or less to 200 kDa or greater, or from 5 kDa or less to 200 kDa or greater, or from 10 kDa or less to 250 kDa or greater, or from 5 kDa or less to 250 kDa or greater.
[0191]In some aspects of the invention, a pre-labeled protein standard set of the invention includes three or more labeled proteins, in which a first and a second protein of the three or more labeled proteins differ from one another by the same molecular weight increment as a second and third protein of the set. In some embodiments, the molecular weight increment, +/-1 kDa, is a multiple of a value between 5 kDa, a multiple of a value between 10 kDa, a multiple of a value between 20 kDa, or a multiple of 50 kDa. In some preferred embodiments, a pre-labeled protein standard set of the invention includes four or more labeled proteins, in which at least four of the four or more labeled proteins differ from one another by a multiple of (plus or minus 1.0 kDa) 10 kDa. In some preferred embodiments, a pre-labeled protein standard set of the invention includes five or more labeled proteins, in which at least five of the five or more labeled proteins differ from one another by a multiple of 10 kDa. In some preferred embodiments, a pre-labeled protein standard set of the invention includes five or more labeled proteins, in which at least 40% of the five or more labeled proteins differ from one another by a multiple of 10 kDa.
[0192]A pre-labeled protein of a standard set of the invention can be made by recombinant methods. Protein standards can be produced in cell culture and purified for selective labeling on one or more target nucleic acids. In some embodiments, the proteins standards have amino acid tag sequences, such as amino acid tags that can be used to purify the proteins. An exemplary amino acid tag is a His tag. Proteins made by recombinant methods can be based on the sequences of naturally-occurring proteins, or can have synthetically designed sequences.
[0193]In some aspects, a pre-labeled protein standard set can include one or more proteins not made by recombinant methods. Labeled proteins of a pre-labeled protein standard set isolated from natural sources, such as organisms, cells, or media, can be enzymatically or chemically modified, such as by addition of chemical protecting groups, or fragmentation by chemical or enzymatic cleavage, or can be unmodified. In some aspects, a pre-labeled protein standard set can include one or more proteins made, at least in part, by synthetic methods, such as chemical synthesis.
[0194]A pre-labeled protein standard set can include one or more proteins that is not selectively labeled. Labeled proteins of a pre-labeled protein standard set on the invention that are not selectively labeled can be recombinant proteins or proteins isolated from cells, tissues, organisms, biological samples, or media. Proteins can also be made wholly or partly using chemical synthesis.
[0195]The invention also includes a set of pre-labeled protein standards that comprises a plurality of labeled proteins, in which one or more of the labeled proteins is selectively labeled on a first amino acid, in which the plurality of labeled proteins are provided in one or more solutions. In preferred embodiments, a pre-labeled protein standard set provided in solution form comprises at least five labeled proteins, in which two, three, four, or five of the labeled proteins are labeled on cysteine and lack lysine. The invention also includes a pre-labeled protein standard set provided in solution form comprises at least 12 labeled proteins, in which the labeled proteins span a molecular weight range of from 10 kDa or less to 100 kDa or greater, in which the electrophoretic migration of each of the pre-labeled protein standards having a molecular weight of 5 kDa or greater is within 5% of the electrophoretic migration of each of the same five or more protein standards in unlabeled form, calculated from the same acrylamide gel. A solution can include one or more buffers, reducing agents, chelators, alcohols, detergents, or dyes.
[0196]Also provided herein is a set of unlabeled protein standards that includes at least two proteins that comprises one or more copies of a sequence derived from a naturally-occurring protein, in which the protein lacks residues of a first amino acid. In some preferred embodiments, the set of unlabeled protein standards comprises two or more proteins that comprise two or more copies of a sequence derived from a naturally-occurring protein, in which the two or more labeled proteins lack lysine residues. In some preferred embodiments, the set of pre-labeled protein standards comprises three or more, four or more, or five or more, six or more seven or more, eight or more, nine or more, ten or more, eleven or more, or twelve or more labeled protein standards in which two or more, three or more, four or more, five or more of the proteins lack lysine and comprise two or more copies of a sequence derived from a naturally-occurring protein. The standards can be labeled with two, three, four, or more visually distinguishable dyes. The standards can span a molecular weight range of from less than 10 kDa to greater than 100 kDa, or from less than 5 kDa to greater than 250 kDa. The standards can have two or more, three or more, four or more, five or more, or six or more protein standards that differ by an increment that is a multiple of 10 kDa (plus or minus 1 kDa). In some embodiments, the ratios of cysteine residues to molecule weight for the two or more, three or more, four or more, five or more proteins that lack lysine do not vary by more than 5%. In some embodiments, each of the two or more, three or more, four or more, five or more proteins that lack lysine have between one and ten, between two and seven, or between three and five cysteine residues per 10 kDa.
Methods of Using a Pre-Labeled Standard Set to Determine Molecular Weight of a Protein
[0197]The invention also includes methods for separating two or more protein standards of a set of pre-labeled protein standards, in which the pre-labeled protein standard set includes at least one protein that is selectively labeled on a first amino acid and is depleted in residues of a second amino acid. In some embodiments of the method, the one or more selectively labeled protein standards The method includes applying the pre-labeled protein standard set to an electrophoresis gel, applying an electric field across the gel, and separating two or more protein standards of the pre-labeled protein standard set. The two or more protein standards are separated such that their bands do not overlap. In preferred embodiments, all of the protein standards of the pre-labeled standard set are separated such that the bands do not overlap and such that the width of the bands on a gel of each of the electrophoresed proteins of the set having a molecular weight of 10 kDa or greater does not vary by more than 2-fold. In preferred embodiments, all of the protein standards of the pre-labeled standard set are separated such that the bands do not overlap the width of the bands on a gel of each of the electrophoresed proteins of the set having a molecular weight of 10 kDa or greater does not vary by more than 2-fold and the band intensities of the proteins of the pre-labeled protein standard set having molecular weights of 10 kDa or greater does not vary by more than 2.5 fold. In preferred embodiments, protein standards of the prelabeled standard set having molecular weights of 10 kDa or greater migrate within 5% of the distance of the that the same protein standards in unlabeled form migrate.
[0198]In a further aspect, methods are provided for characterizing one or more sample proteins using a pre-labeled protein standard set provided herein. The method includes electrophoresing one or more proteins and at least one prelabeled protein standard set as described herein in a gel; and comparing the migration of the one or more proteins with the migration of least one protein standard of the pre-labeled standard set. In some preferred embodiments, the method further comprises determining the molecular weight of the one or more sample proteins.
[0199]For example, the method includes electrophoresing a sample that includes one or more proteins in a first lane of a gel and electrophoresing a pre-labeled protein standard set that comprises at least two labeled proteins that are selectively labeled on a first amino acid in a second lane of the gel, determining the migration distance of at least two of the two or more labeled proteins of the standard, determining the migration distance of at least one of the one or more sample proteins, and calculating the molecular weight of the at least one sample protein based on the migration distance and molecular weights of the at least two labeled proteins of the standard. The method can use point-to point calibration or can compare migration distances by generating a curve based on migration distance versus molecular weight (or log of molecular weight), for example using the least squares method.
[0200]In preferred embodiments, all of the protein standards of the pre-labeled standard set are separated from one another such that the bands do not overlap and such that the widths of the bands on a gel of each of the electrophoresed proteins of the set having a molecular weight of 10 kDa or greater do not vary by more than 2-fold. In preferred embodiments, all of the protein standards of the pre-labeled standard set are separated such that the bands do not overlap the widths of the bands on a gel of each of the electrophoresed proteins of the set having a molecular weight of 10 kDa or greater do not vary by more than 2-fold and the band intensities of the proteins of the pre-labeled protein standard set having molecular weights of 10 kDa or greater do not vary by more than 2.5 fold. In preferred embodiments, protein standards of the prelabeled standard set having molecular weights of 10 kDa or greater migrate within 5% of the distance of the that the same protein standards in unlabeled form migrate.
Pre-Labeled Protein Standard Kits
[0201]The invention also includes kits that include the described pre-labeled protein standard sets, and further comprise one or more of one or more buffers, loading dyes, reducing agents, unlabeled protein standards, blotting membranes, gel cassettes, pre-cast gels, or electrophoresis buffers. The components of the kit can in one or more containers, and two or more of the components of the kit can be provided in a common package (such as, for example, a box, rack, or jar). The kit can also include instructions for use, or instructions for accessing protocols for use of the kit or its components via the internet.
[0202]The set of pre-labeled protein standards of the kit can be provided as lyophilized solids, or in solution in liquid or frozen form. A solution comprising one or more labeled protein standards of a set can include one or more buffers, reducing agents, chelators, alcohols, detergents, or dyes. The set of pre-labeled protein standards of the kit can include at five, six, seven, eight, nine, ten, eleven, twelve, or more labeled protein standards that are provided as one or more mixtures of two or more labeled standards. In some embodiments, all of the proteins of a pre-labeled protein standard set are provided in a single mixture (which can be provided in one or more aliquots) in a kit. The proteins of a pre-labeled protein standard set provided in a kit preferably span a molecular weight range of from 10 kDa or less to 100 kDa or more, and can span a molecular weight range of from 5 kDa or less to 250 kDa or more.
[0203]In one embodiment of a kit, a pre-labeled standard set provided in a kit comprises a plurality of labeled proteins, in which one or more of the labeled proteins is selectively labeled on a first amino acid and lacks a second amino acid that is capable of reacting with a dye used to label the protein.
[0204]In some preferred embodiments, a pre-labeled protein standard set provided in a kit comprises at least five labeled proteins, in which two, three, four, or five of the labeled proteins are labeled on cysteine and lack lysine. The invention also includes a kit that comprises a pre-labeled protein standard set that comprises at least 10 labeled proteins, in which the labeled proteins span a molecular weight range of from 10 kDa or less to 100 kDa or greater, in which the electrophoretic migration of each of the pre-labeled protein standards having a molecular weight of 5 kDa or greater is within 5% of the electrophoretic migration of each of the same protein standards in unlabeled form, calculated from the same acrylamide gel. The invention also includes a kit that comprises a pre-labeled protein standard set that comprises at least 12 labeled proteins, in which the labeled proteins span a molecular weight range of from 5 kDa or less to 260 kDa or greater, in which the electrophoretic migration of each of the pre-labeled protein standards having a molecular weight of 5 kDa or greater is within 5% of the electrophoretic migration of each of the same protein standards in unlabeled form, calculated from the same acrylamide gel.
[0205]In some embodiments, a pre-labeled protein standard set provided in a kit includes two or more proteins labeled on a first amino acid, in which the ratios of the number of residues of the first amino acid to molecular weight of at least two of the two or more labeled proteins are within 5% of one another, in some embodiments within 2.5% of one another. In some preferred embodiments of a pre-labeled protein standard set provided in a kit, at least two proteins selectively labeled on a first amino acid have between two and seven, or between three and five residues of a first amino acid, such as between 3.5 and 4.5 residues of a first amino acid per 10 kDa. In some preferred embodiments of a pre-labeled protein standard set provided in a kit, at least five proteins of the set that are selectively labeled on a first amino acid have between three and five residues of a first amino acid, such as between 3.5 and 4.5 residues of a first amino acid per 10 kDa. In some preferred embodiments, a pre-labeled protein standard set provided in a kit comprises at least five labeled proteins, in which two, three, four, or five of the labeled proteins are labeled on cysteine and lack lysine, and the two, three, four, or five labeled proteins have a ratios of cysteine residues to molecular weight that are within 5% of one another. In these embodiments, the two, three, four, or five labeled proteins can have between two and seven, or between two and five, cysteine residues per 10 kDa.
[0206]Also provided herein is kit comprising a set of pre-labeled protein standards that includes at least one labeled protein that comprises two or more copies of a sequence derived from a naturally-occurring protein, in which the at least one labeled protein lacks lysine residues and is labeled on at least one cysteine residue. In some preferred embodiments, the set of pre-labeled protein standards comprises two or more labeled proteins that comprise two or more copies of a sequence derived from a naturally-occurring protein, in which the two or more labeled proteins lack lysine residues and are labeled on at least one cysteine residue. In some preferred embodiments, the set of pre-labeled protein standards comprises three or more, four or more, or five or more, six or more seven or more, eight or more, nine or more, ten or more, eleven or more, or twelve or more labeled protein standards in which two or more, three or more, four or more, five or more of the cysteine-labeled proteins that lack lysine comprise two or more copies of a sequence derived from a naturally-occurring protein. The standards can be labeled with two, three, four, or more visually distinguishable dyes. The standards can span a molecular weight range of from less than 10 kDa to greater than 100 kDa, or from less than 5 kDa to greater than 250 kDa. The standards can have two or more, three or more, four or more, five or more, or six or more protein standards that differ by an increment that is a multiple of 10 kDa (plus or minus 1 kDa). In preferred embodiments, all of the protein pre-labeled standards of the set can migrate within 5% of the migration of the same proteins in unlabeled form. In preferred embodiments, the ratios of cysteine residues to molecule weight for the two or more, three or more, four or more, five or more cys-labeled proteins that lack lysine do not vary by more than 5%. In preferred embodiments, each of the two or more, three or more, four or more, five or more cys-labeled proteins that lack lysine have between one and ten, between two and seven, or between three and five cysteine residues per 10 kDa.
[0207]In related embodiments, a pre-labeled protein standard set of the invention includes three or more labeled proteins, in which a first and a second protein of the three or more labeled proteins differ from one another by the same molecular weight increment as a second and third protein of the set. In some embodiments, the molecular weight increment is, when rounded to the nearest 1 kDa, a multiple of 5 kDa, a multiple of 10 kDa, a multiple of 20 kDa, or a multiple of 50 kDa. In some preferred embodiments, a pre-labeled protein standard set of the invention includes five or more labeled proteins, in which at least five of the five or more labeled proteins differ from one another by a multiple of 10 kDa. In some preferred embodiments, a pre-labeled protein standard set provided in a kit comprises at least five labeled proteins, in which two, three, four, or five of the labeled proteins are labeled on cysteine and lack lysine, and at least three, at least four, or at least five of the labeled proteins of the set differ in molecular weight increments by a multiple of 10 kDa (plus or minus 1 kDa).
[0208]In another aspect, the invention provides methods of providing a set of pre-labeled protein standards to a customer, in which the set of pre-labeled protein standards includes any of the pre-labeled standard sets and kits disclosed herein. In one embodiment, the method includes: providing a pre-labeled protein standard set to a customer, in which at least one of the labeled proteins of the standard set is selectively labeled on a first amino acid, in exchange for revenue. In another embodiment, the method includes: providing a pre-labeled protein standard set to a customer, in which the pre-labeled protein standard set comprises from five to twelve labeled proteins, and at least five of the labeled protein are labeled on cysteine and lack lysine residues. In another embodiment, the method includes: providing a pre-labeled protein standard set to a customer, in which two or more of the labeled proteins of the standard set is selectively labeled on a first amino acid and at least two of the two or more selectively labeled proteins have a constant ratio of a first amino acid to molecular weight, in exchange for revenue. In another embodiment, the method includes: providing a pre-labeled protein standard set to a customer, in which the pre-labeled protein standard set comprises from five to twelve labeled proteins, and at least five of the labeled protein are labeled on cysteine and lack lysine residues, and the at least five labeled protein have the same ratio of cysteine residues to molecular weight. In another embodiment, the method includes: providing a pre-labeled protein standard set to a customer, in which the pre-labeled protein standard set includes 12 or more labeled proteins, in which the migration of each of the labeled protein standards having a molecular weight of 5 kDa or greater is within 5% of the migration of each of the five or more protein standards in unlabeled form on the same acrylamide gels, in exchange for revenue. In another embodiment, the method includes: providing a pre-labeled protein standard set to a customer, in which the pre-labeled protein standard set comprises twelve labeled proteins, in which at least five of the twelve labeled proteins are labeled on cysteine and lack lysine residues, and in which the electrophoretic migration of each of the twelve labeled protein standards is the same as the electrophoretic migration of the same protein standard in unlabeled form on the same acrylamide gel.
[0209]The following examples are intended to illustrate but not limit the invention.
EXAMPLE 1
Sharp Molecular Weight Marker Expression Plasmids: 30, 40, and 50 kDa Proteins
[0210]Expression plasmids for the 30, 40, and 50 kDa proteins were made using pTrcBH 60 kd, a construct containing a synthetically derived open reading frame (ORF) consisting of six tandem E. coli thioredoxin (Thio) segments. BlueHeron® Biotechnology (Bothell, Wash., USA) was contracted to synthesize the 1595 bp ORF according to specifications that would allow for optimal protein-dye labeling.
[0211]FIG. 1A aligns the truncated thioredoxin ORF of clone pTrxfusprl10A (see U.S. Pat. No. 6,703,484, herein incorporated by reference in its entirety having: 1) 23 amino acids removed from the carboxy terminus, 2) a substitution of glu for val at the last Thio (86th) codon position, and 3) 6 C-terminal histidines added to the C terminus, with the Thio ORF (top row of FIG. 1B) that was modified to contain 4 cysteine (C) and no lysine (K) amino acids. Two additional cysteines were added to the ORF by codon modification of serine residues (S) at positions 2 and 12. All 7 lysine (K) amino acids were changed to arginine (R) at positions 4, 19, 52, 70, 83 and methionine (M) at position 36 to favor the binding of the dye molecules to cysteine rather than lysine. Cysteine and methionine at positions 35 and 37 were replaced with arginine and cysteine to increase the distance between cysteine residues and minimize the potential steric hindrance created by two dye molecules binding to cysteines residues at positions 34 and 37. Isoleucines at positions 23 and 45 were changed to arginine to decrease the protein's predicted hydrophobicity.
[0212]The Thio ORF of 279 bp was truncated to meet the molecular weight requirements of the final product. In creating a six Thio repeat construct, the first of six Thio repeats of pTrcBH 60 kd was set at 208 bp (providing a translation product of 7.7 kd) and the remaining five identical repeats were set at 258 bp (each providing a translation product of 9.8 kd). Approximately every 18th amino acid's 3rd base codon wobbled to minimize repeats when the construct was fully assembled.
[0213]As shown by the diagram of FIG. 2A the six assembled Thio repeats were separated by five unique restriction sites. The 5' end of the six Thio repeat ORF contained a Bgl II site and the 3' end, containing the five unique restriction sites followed by a ten HIS sequence and capped with a Pme I site. This design allowed for the subcloning of this ORF, referred to a BH6mer ORF (SEQ ID NO:13, FIG. 2B), into a pTrc expression vector (Invitrogen, Carlsbad, Calif., USA) using BamH1 and Pme1 restriction sites, creating an expression plasmid from which an approximately 60 kd translation product could be made, with the flexibility of generating expression plasmids for synthesizing translation products of approximately 10, 20, 30, 40, or 50 kd from the same vector, depending on which of the five unique restriction site enzyme was employed to digest the plasmid before re-closing it to make a shorter construct.
[0214]FIG. 3A shows the map of the pTrc BH 60 kDa cloning construct used to generate the lower molecular weight pTrc BH 30 kDa construct (shown in FIG. 4A), pTrc BH 40 kDa construct (shown in FIG. 5A), and pTrc BH 50 kDa construct (shown in FIG. 6A).
[0215]The sequence-verified Thio repeat ORF insert (BH6mer ORF) from BlueHeron® Biotechnology (FIG. 2B, SEQ ID NO:13) was cut out of their pUC-minus cloning vector by sequential digests using PmeI followed by Bgl II. The six Thio insert (1595 bp) was gel purified and eluted using a S.N.A.P® resin mini column (Invitrogen, Carlsbad, Calif., USA) and centrifugation at 14,000 rpm for 10 minutes at room temperature and ligated to a modified pTrc LacZ-Flash vector.
[0216]The pTrc LacZ-Flash expression vector that includes a LacZ ORF with a C-terminal lumio sequence and a 10 his tag, a trp/lac inducible promoter and sequences for enhancing expression of eukaryotic genes in E. coli. It was mutagenized by restriction digestion and ligation to delete the single NcoI site to allow for in-frame translation of the BH6mer ORF. The modified pTrc expression vector was digested with BamHI and PmeI and the 4285 bp vector fragment was gel purified.
[0217]The BH6mer ORF was ligated into the digested pTrc vector backbone via BamHI-PmeI to generate the pTrc BH 60 kd expression construct having the insert shown in FIG. 2A. Restriction digest screening using BamHI and EcoR I identified a positive clone and protein expression screening in BL21 DE3 STAR verified the restriction digest results.
[0218]The amino acid composition of the pTrc BH 60 kd protein determined by DNA sequencing of the construct showed a valine (V) residue capping the C-terminal 10 HIS sequence (FIG. 3B; SEQ ID NO:14). The valine capped HIS sequence originated from the pTrc LacZ-Flash vector within the Pme I site. The presence of this valine on the end of the 10 HIS tag did not affect Ni-NTA purification of the synthesized protein.
[0219]Additional pTrc BH expression clones were obtained by restriction digests using one of the five unique sites depicted in FIG. 2A. The map of pTrc BH 30 kd and the sequence of the 30 kDa ORF encoded by the insert (SEQ ID NO:15) is shown in FIG. 4. The map of pTrc BH 40 kd and the sequence of the 40 kDa ORF encoded by the insert (SEQ ID NO:16) is shown in FIG. 5. The map of pTrc BH 50 kd and the sequence of the 50 kDa ORF encoded by the insert (SEQ ID NO:17) is shown in FIG. 6. Clones were screened by colony PCR to identify positive expression constructs using the following primers: #24 pTrCHisFOR: GAGGTATATATTAATGTATCG (SEQ ID NO:18) and #12 pBAD_Rev: GATTTAATCTGTATCAGG (SEQ ID NO:19). Protein expression screens in BL21 DE3 STAR were preformed to validate PCR screen screening results.
EXAMPLE 2
Sharp Molecular Weight Marker Expression Plasmids: 110, 160, and 260 kd Proteins
[0220]Expression constructs encoding 100, 150, and 250 kd proteins containing multimers of the BH6mer ORF, which contained 4 cys and 0 lys residues per 10 kd were made using insert fragments of the pTrc BH 60 kDa expression construct of Example 1 generated by PCR.
Synthesis of 50 kd PCR Inserts (1314 bp)
[0221]Using the pTrc BH 60 kDa expression construct of Example 1 as the PCR template, several 50 kDa inserts were generated using Platinum® PCR Supermix High Fidelity PCR mix (Invitrogen; Carlsbad, Calif.) that contained Taq DNA polymerase, Pyrococcus species GB-D thermostable polymerase, Platinum® anti-Taq polymerase antibody, 66 mM Tris-SO4 (pH 8.9), 19.8 mM (NH4)2SO4; 2.4 mM MgSO4; 220 μM dNTPs; and stabilizers; with the following primer sets:
TABLE-US-00002 50.1_F: (SEQ ID NO:20) CCGGAGATCTATGTGTGATCGTATTATTCA and 50.1_R: (SEQ ID NO:21) CCGGCTCGAGTTCGCCGTTACGGAAAAGCA; 50.2_F: (SEQ ID NO:22) CCGGCTCGAGATGTGTGATCGTATTATTCATCTGAC and 50.2_R: (SEQ ID NO:23) CCGGCCTAGGTTCGCCGTTACGGAAAAGCA, or 50.2_10HIS-Pme_R: (SEQ ID NO:24) GTTTAAACGTGATGATGATGGTGGTG GTGGTGG TGGTGTTCGCCGTTA CGGAAAAGCAGAAG; 50.3_F: (SEQ ID NO:25) CCGGCCTAGGATGTGTGATCGTATTATTCATCTGAC, and 50.3_R: (SEQ ID NO:26) CCGGCGGCCGTTCGCCGTTACGGAAAAGCA, or 50.3_10HIS-Pme_R: (SEQ ID NO:27) GTTTAAACGTGATGATGATGGTGGTGGTGGTGGTGGTGTTCGCCGTTACG GAAAAGCAGAAG; 50.4_F: (SEQ ID NO:28) CCGGCGGCCGATGTGTGATCGTATTATTCAT, and 50.4_10HIS-Pme_R: (SEQ ID NO:29) GTTTAAACGTGATGATGATGGTGGTGGTGGTGGTGGT GTTCGCCGTTAC GGAAAAGCAGAAG
[0222]The 1314 bp inserts (50 kDa) were gel purified on a 1.2% E-Gel®. The PCR inserts were TA cloned into pCR2.1 (Invitrogen; Carlsbad, Calif.) using the manufacturer's protocol. Primer design allowed for each 50 kd TA clone to have unique sequence ends that facilitated vector construction as shown in Table 2.
TABLE-US-00003 TABLE 2 50 kd Inserts used for High Molecular Weight Marker Constructs Insert Name Insert Configuration TA 50.1 BgL II-50 kd-Xho I TA 50.2 Xho I-50 kd-Avr II TA 50.2-10HIS-PmeI Xho I-50 kd-10HIS-PmeI TA 50.3 AvrII-50 kd-EagI TA 50.3-10HIS-PmeI AvrII-50 kd-10HIS-PmeI TA 50.4-10HIS-PmeI EagI-50kd-10HIS-PmeI MM 50 kd XhoI-SpeI-XbaI-BgLII-50 kd-NheI-BamHI-PstI
[0223]White colonies were selected for colony PCR screening using the specific primer sets used in the cloning. The sequences of TA inserts of the 50.2 insert of clone 50.2_B3, the 50.3 insert of clone 50.3_C14, the 50.4 insert of clone 50.4-10HIS-PmeI_C4, and the MM 50 kd insert of an MM 50 kd clone were confirmed using the primers in Table 3.
TABLE-US-00004 TABLE 3 Sequencing Primers used to Confirm 50 kd Inserts Primer Sequence TA50kd_1F GTGCGGTCCACGTATGTG (SEQ ID NO:30) TA50kd_2F GGCGCGTCTCGTCGAC (SEQ ID NO:31) TA50kd_2R ACTCTGCCCAGAAGTCGAC (SEQ ID NO:32) TA50kd_3F CGAAACCGGTATGTGCG (SEQ ID NO:33) TA50kd_3R CGATCGCACATACCGG (SEQ ID NO:34) T7#6 TAATACGACTCACTATAGGG (SEQ ID NO:35) PCRII200F#738 CACACAGGAAACAGCTATGA (SEQ ID NO:36)
[0224]All of the sequenced clones contained the identical 50 kd-encoding 1314 bp sequence of SEQ ID NO:37 (FIG. 7).
[0225]Assembly of pTrc 50 kDa Base Vector, and pTrc 110 kDa, pTrc 160 kDa, and pTrc 260 kDa Expression Vectors
pTrc 50 kDa Base Vector:
[0226]TA clone 50.1_clone 2D was digested with BgL II and Not I (site from the pCR2.1 vector) to remove the 50 kDa insert. The fragment was gel purified. The modified pTrc LacZ-Flash vector was digested with BamHI-Not I and the gel purified (4377 bp) vector was ligated with the TA 50.1--2D insert.
[0227]A positive clone was identified by colony PCR using the 50.1 forward primer (SEQ ID NO:20) and 50.1 reverse primer (SEQ ID NO:21). This clone, labeled pTrc 50.1 D3 was the base construct used in subsequent subclonings for construction of the pTrc 110 kDa, pTrc 160 kDa, and pTrc 260 kDa expression vectors.
pTrc 110 kd Expression Vector:
[0228]TA clone 50.2-10HIS-PmeI clone B6 was digested with XhoI and PmeI. The gel purified insert was subcloned into pTrc 50.1 D3 which had been also digested with XhoI and PmeI. XhoI and PmeI restriction digest screening identified a positive clone that was later confirmed by protein expression screening.
[0229]The expressed protein had a molecular weight that was closer to 110 kDa than to the expected 100 kDa. It is believed that during the preparation of the fragments one of the presumed 50 kDa subcloned fragments (the first or the second) was a 60 kDa Thio repeat fragment instead of a 50 kDa Thio repeat fragment. Mass spectrometry analysis of the actual molecular weight of the expressed protein revealed that it was 10 kDa larger than expected (Table 4). The expression clone was labeled pTrc 50.1-2 Pme, Clone B6-9 and renamed pTrc 110 kd (FIG. 8A). The sequence of the insert was not directly determined. The predicted sequences based on the cloned fragments is provided as SEQ ID NO:38 in FIG. 8B).
pTrc 160 kd Expression Vector:
[0230]TA clone 50.2 clone B3 was digested with XhoI and Not I (site from pCR2.1) to remove the 50 kDa insert. The pTrc 50.1 D3 vector was digested with XhoI and Not I and the gel purified vector was ligated with the 50.2_B3 gel purified insert. A positive clone was identified by restriction digest screening using XhoI-AvrII and was labeled pTrc1-2 C6.
[0231]The pTrc1-2 C6 vector, containing two 50 kd inserts, was digested with Avr II and PmeI. The gel purified vector was ligated with TA clone 50.3-HIS-Pme I insert that had been digested with AvrII and PmeI and gel purified. A positive clone was identified by restriction digest screening using Avr II-PmeI and later confirmed by protein expression screening. In this case, the expressed protein had a molecular weight that was closer to 160 kDa than to the expected 150 kDa. It is believed that during the preparation of the fragments one of the presumed 50 kDa subcloned fragments was a 60 kDa Thio repeat fragment instead of a 50 kDa Thio repeat fragment. Mass spectrometry analysis of the actual molecular weight of the expressed protein revealed that it was 10 kDa larger than expected (Table 4). The expression clone was labeled pTrc1,2,3 Pme and renamed: pTrc 160 kd (FIG. 9A). The sequence of the insert was not directly determined. The predicted sequences based on the cloned fragments is provided as SEQ ID NO:39 in FIG. 9B).
pTrc 260 kd Expression Vector:
[0232]A 260 kDa protein expression vector, pTrc 160+LacZ, was also constructed. Using the unique restriction site (Avr II), located between 50 kDa Thio repeat fragments 2 and 3 in the pTrc 160 kDa protein construct (FIG. 9), a truncated LacZ gene encoding a 100 kDa polypeptide (SEQ ID NO:40; FIG. 10) was cloned into the AvrII site.
[0233]The 260 kDa protein had an estimated mass of 253,624 daltons. The protein contained 73 cysteines and 19 lysine amino acids. The sequence of the insert was not directly determined. The predicted sequences based on the cloned fragments is provided as SEQ ID NO:41 in FIG. 11B).
[0234]The LacZ gene was generated with Platinum® PCR Supermix High Fidelity PCR mix (Invitrogen; Carlsbad, Calif.) using primers capped with Avr II restriction sites. The resulting PCR product was Topo cloned into the pCR®-Blunt cloning vector (Invitrogen, Carlsbad, Calif., USA) using the Zero Blunt® kit (Invitrogen, Carlsbad, Calif., USA).
[0235]The truncated LacZ ORF was excised from the cloning vector with Avr II digestion and the fragment was gel purified. The pTrc 160 kDa construct was linearized with AvrII and gel purified.
[0236]The truncated LacZ insert was ligated into a non-alkaline phosphatase treated pTrc 160 kDa vector. The ligation reaction was transformed into One Shot® Top 10 competent bacterial cells (Invitrogen, Carlsbad Calif., USA) and the resulting colonies were PCR screened for the LacZ gene. PCR colony screening identified 11/80 clones containing the LacZ insert and expression screening identified 5/11 clones having the LacZ insert in the correct orientation. The pTrc 160+LacZ clone BI in BL 21 DE3 was expressed in 1.0 L of BRM-Amp, 30° C., 18 hrs, uninduced, to verify expression performance. This clone was subsequently designated pTrc 260 kDa (FIG. 11A).
[0237]To test for expression of proteins, expression plasmids were transformed into competent BL21-DE3 cells. The cells were grown in LB media with 100 ug/ml Ampicillin at 37° C. IPTG was added to 1 mM when the OD600 reached 0.4-0.6 and the cells were incubated at 37° C. for an additional 4-6 hours.
[0238]After the expression period 1 ml of the cell cultures were centrifuged at 5000×g for 5 minutes. The liquid fraction was discarded and 100 μl of BugBuster® HT protein extraction reagent (Novagen, Madison, Wis., USA) with 25 ug/ml lysozyme was added to the cells. The sample was vortexed to resuspend the cells and incubated for 10 minutes at room temperature. 50 μl of the lysate was transferred to a separate tube. Another 50 ul of the lysed bacterial sample was centrifuged at 10,000×g for 5 minutes. The liquid fraction was discarded and the pellet (insoluble fraction) was resuspended in 50 μl of 1×LDS Sample buffer.
[0239]5 μl of 4×LDS and 2 μl NuPAGE reducing reagent were added to 15 μl of the whole lysate and to 15 μl of insoluble fraction. The samples were incubated for 10 minutes at 70° C. 10 μl of each sample were loaded on a 4-12% NuPAGE® gel and run with 1×MES running buffer at 200V for 37 minutes. The gel was stained with SimplyBlue® SafeStain protein stain using the microwave protocol to visualized the expressed proteins.
EXAMPLE 3
Production of Recombinant Proteins
[0240]The following procedures were used for the production of recombinant proteins for use as molecular weight standards.
[0241]30, 40, 50 and 110 kDa (no-lysine (NL)) proteins
[0242]Reagents [0243]BugBuster® HT protein extraction reagent (Novagen, Madison, Wis., USA) [0244]Freshly prepared 25 mg/ml lysozyme in ultrapure water [0245]Induced 50 ml cell cultures (after reaching an O.D. of 0.5 in that contains rich media [24 g/L yeast extract, 12 g/L tryptone, 0.05% glucose, 1 mM MgSO4, 50 mM KH2PO4, 50 mM K2HPO4, 10 mM (NH4)2--SO4, and 1% glycerol], lactose is added to 1 mM, and the culture is incubated overnight at a temperature of 32 degrees C. or 37 degrees C., or as low as 30 degrees C.)
[0246]Large scale cultures can be grown in a 7 L fermentor (e.g., an Applikon fermentor) through which air is bubbled. Protein molecular weight standards were produced in large quantity by inoculating a 2.8 L non-baffled seed flask of approximately 1 liter of rich media with a freshly transformed (less than one week old) colony containing the expression plasmid. (Rich media per liter: 12 grams of tryptone, 24 grams of yeast extract dissolved in distilled water to a final volume of 1 liter is autoclaved, and after cooling to approximately 30 degrees C., 10 mls of 10 mg/ml ampicillin, 50 mls of 20×NPS, 10 mls of 5052 solution, and 1 ml of 1 molar Magnesium Sulfate are added. 20×NPS is made by adding 66 g ammonium sulfate; 136 g potassium phosphate, monobasic; and 142 g potassium phosphate, dibasic, per liter distilled water. 5052 solution is made by adding 500 grams of glycerol and 50 grams of glucose per liter of distilled water. 20×NPS and 5052 solutions are filter sterilized using micron filters.) The seed flask is incubated with shaking (250 rpm) at 30 degrees C. until the OD is between 1.0 and 3.0 (approximately 7-9 hours).
[0247]150 mls of the seed flask culture is then transferred to a 7 liter fermentor that contains 5 liters of rich media made as for the seed culture. The fementor is incubated with aeration parameters at 1.25 lpm air, 500 rpm agitation, and the pH is controlled to 6.8 using KOH or 5 M H3PO4. Incubation is at 30 degrees C. for approximately 1.5 to 2 hours, or until the OD reaches 0.5 to 1. At this time lactose is added to the culture to a final concentration of between 0.05% and 0.5%. For example, 50 mls of a solution of 20% lactose is added to the 5 L culture for a final concentration of 0.2% lactose.
[0248]The cells are harvested at early stationary phase, when two consecutive hourly readings of less than 0.5 OD change. This generally occurs 14-17 hours after inoculation. The final OD is generally 10 or greater.
[0249]Protein Isolation [0250]8M urea, 20 mM phosphate, 500 mM NaCl pH=7.8 [0251]Ni-NTA resin [0252]8M urea, 20 mM phosphate, 500 mM NaCl pH=6 [0253]8M urea, 20 mM phosphate, 500 mM NaCl pH=4 [0254]10N NaOH
[0255]Materials and Equipment [0256]50 ml centrifuge tubes [0257]Centrifuge capable of obtaining 10,000×g force
[0258]Protein Extraction [0259]50 ml cell culture is centrifuged at 5000×g for 10 minutes [0260]The cell media is discarded and 2.5 ml BugBuster® HT protein extraction reagent (Novagen, Madison, Wis., USA) including 25 μl of 5 mg/ml lysozyme are added to the cell paste [0261]The cells are re-suspended in the lysis reagent by vortexing intermittently for 30 minutes at room temperature [0262]The lysed sample is centrifuged for 10 minutes at 8,000×g. [0263]The soluble fraction is discarded [0264]4 ml 8M urea, 20 mM phosphate, 500 mM NaCl pH=7.8 is added to the pellet. [0265]The sample is vortexed for 10-15 seconds to disperse the pellet and then immediately mixed using a Polytron mixer. [0266]If the sample looks clear after the mixing with the Polytron centrifugation is performed. Otherwise the sample is warmed at 70° C. for 5 minutes to facilitate the solubilization of protein prior to centrifugation. [0267]The sample is left to cool down to room temperature [0268]The sample is centrifuged for 5 minutes at 5,000×g to pellet cell debris
NTA Purification
[0269]The solubilized protein is loaded on a 10 ml Ni-NTA column equilibrated in 8M urea, 20 mM phosphate, 500 mM NaCl pH=7.8. The column is attached to a stand and the liquid is drained from the column. The column is plugged with a cap and 4 ml 8M urea, 20 mM phosphate, 500 mM NaCl pH=7.8 are added to the column. The column is incubated on the shaker for 2 minutes and then the wash is drained from the column. The pH 7.8 wash process is repeated 1 more time.4 ml of 8M urea, 20 mM phosphate, 500 mM NaCl pH=6 are added to the column and the column is incubated for 2 minutes on the shaker. The wash solution is discarded and the pH 6 wash process is repeated 1 more time. The bound protein is eluted with addition of 5 ml 8M urea, 20 mM phosphate, 500 mM NaCl pH=4 to the top of the column and collecting 1 ml fractions. The collected fractions are analyzed by electrohoresis. The fractions with the purified proteins are pooled together and the pH is adjusted to 7.5-8 with NaOH.
Protein Concentration
[0270]14 ml 60% TCA is added to 30 ml protein solution obtained from the Ni-NTA purification add and mixed well. The protein solution plus TCA is incubated at 4° C. for 1-2 hours and then centrifuged at 8,000×g for 10 minutes at 4° C. The liquid is discarded and 30 ml of ultrapure H2O is added and mixed well. The protein is centrifuged at 8000×g for 10 minutes and liquid is discarded taking care not to discard the protein pellet. The H2O wash is repeated, and then 300 μl of 50 mM Tris, 1% SDS pH=8 is added to the pellet. The protein is heated at 70° C. for 10-15 minutes if needed and vortexed to resolubilize the protein.
160 and 260 kDa purification
[0271]Reagents: Complete Protease Inhibitor (Roche Applied Science, Indianapolis, Ind., USA); Freshly prepared 25 mg/ml lysozyme (Calbiochem, San Diego, Calif., USA) in ultrapure water; Induced cell culture as for 30, 40, 50 and 1110 kDa (NL) proteins; Amberlite MB-150 (Sigma-Aldrich); Toyopearl AF Chelate 650M (Tosoh Bioscience, Tokyo, Japan); CHAPS detergent; Urea; 1M Na-phosphate pH=7.8; Imidazole; 5M HCl; Cobalt II chloride.
Preparation of Solutions:
[0272]Conditioning Solution: 8M urea, 20 mM phosphate, 0.5% CHAPS pH=7.8 (2 liters)
[0273]Solubilize 960 g of urea in water. Deionize for 2 or more hours with 10 g/liter Amberlite mixed bed resin. Adjust the volume to 2 liters. Filter through 0.2 or 0.4 um filter. Add 40 ml 1M sodium phosphate pH=7.8. Add 10 grams of CHAPS and mix until solubilized
Elution buffer: 8M urea, 200 mM Imidazole, 0.5% CHAPS pH=7.8
[0274]Solubilize 960 g of urea in water. De-ionize for 2 or more hours with 10 g/liter Amberlite mixed bed resin. Adjust the volume to 2 liters. Filter through 0.2 or 0.4 um filter. Add 27 grams of imidazole. Titrate the pH to 7.8-8 with 5M HCl. Add 10 grams of CHAPS and mix until solubilized.
[0275]Extracting the protein is performed as follows: 10 ml BugBuster® HT protein extraction reagent (Novagen, Madison, Wis., USA) with Complete Protease Inhibitor (Roche Applied Science, Indianapolis, Ind., USA) is added per every 1 g cell paste. One tablet of inhibitor is used for every 50 ml solution. 40 μl of 25 mg/ml lysozyme are added per every 1 gram paste. The cells are re-suspended in the lysis reagent by vortexing. The lysis is performed for 1 hour at room temperature on shaker or rotary mixer. The lysed sample is centrifuged for 10 minutes at 8,000×g. The soluble fraction is discarded. 5 ml of Column Conditioning solution (8M urea, 20 mM phosphate, 0.5% CHAPS pH=7.8) is added for each gram of cell paste. The cell paste is vortexed for 10-20 seconds to break the pellet and the paste is mixed with the Polytron right away. The sample is centrifuged at 8,000×g for 10 minutes to remove any insoluble particles. The solubilized fraction is retained for HIS purification. The purification should be performed the same day the lysate is prepared.
[0276]HIS purification is performed as follows: Toyopearl Chelate 650M resin (Tosoh Bioscience, Tokyo, Japan) is loaded with cobalt II chloride. The resin is washed extensively with water to remove any unbound cobalt The column should be a light pink color after washing with water. The column is washed extensively with Column Conditioning solution (8M urea, 20 mM phosphate, 0.5% CHAPS pH=7.8). The sample is loaded on the column (about 20 ml of sample can be applied to 100 ml column bed volume). The flow rate is stopped and the column is incubated for 1 hour at room temperature. The column is washed until the signal UV 280 nm signal goes to the baseline with Column Conditioning Solution. Protein is eluted with Elution buffer (8M urea, 200 mM Imidazole, 0.5% CHAPS pH=7.8).
[0277]For buffer exchange, a Bio-Gel P-6 column is prepared having 10 column volumes to the sample volume. The column is equilibrated with 50 mM Tris, 1% SDS pH=8.20% SDS is mixed to the sample to a final concentration of 1%. The sample is run through the column and fractions are monitored using 280 nm detection. The first peak is collected as the protein peak. The protein is concentrated to 2-3 mg/ml using 100 kDa MWCO membrane.
Protein Quantitation
[0278]Contaminating bands can interfere with the accurate estimation of protein concentration if total protein concentration in solution is determined. Therefore a gel-based method for protein quantitation is preferred for the molecular weight standard proteins.
[0279]A standard solution of 2 mg/ml Bovine Serum Albumin (BSA) from Pierce Biotechnology (Rockford, Ill., USA) is used to compare band intensities on electrophoresis gels. 1 μl of the 2 mg/ml BSA solution is added to 25 μl of 4×LDS Sample Buffer, 64 μl water and 10 ul NuPAGE® Reducing Reagent (Invitrogen, Carlsbad, Calif., USA). A sample that includes 1 μl of the concentrated molecular weight standard protein is prepared the same way and both samples are incubated for 10 minutes at 70° C. The BSA standard and molecular weight standard protein (5 μl of each) are run side by side on an electrophoresis gel. Multiple standards are preferably compared on the same gel, in which 5 μl of each marker protein sample is loaded between lanes of the BSA standard. The sample concentration is determined visually or using the Alpha Imager 3000 with quantitation software (Alpha Innotech, San Leandro, Calif., USA).
EXAMPLE 5
Insulin b-Chain Purification
[0280]Bovine Insulin consists of two polypeptide chains: Peptide Insulin B chain: theoretical pI: 6.90/Mw (average mass): 3399.93; and Peptide Insulin A chain: theoretical pI: 3.79/Mw (average mass): 2339.65. The bovine insulin b-chain was purified by reduction of bovine pancreas insulin (Sigma-Aldrich, St. Louis, Mo., USA) at denaturing conditions and then separation of the b-chain on an ion exchange column.
[0281]The method used for purification was the following: insulin was solubilized at 5 mg/ml in 8M urea, 50 mM Tris pH=8. 10 ul of 400 mM tributylphosphine (TBP) was added per every ml of solution (to 4 mM final concentration). The solution was heated for 5 minutes at 70° C. with occasional vortexing. The solution became clear and was cooled to room temperature. The sample was loaded on a DEAE ion exchange column equilibrated with 8M urea in 50 mM Na-acetate pH=5.3. The column was washed with 8M urea in 50 mM Na-acetate pH=5.3 for 10 minutes. The b-chain eluted in the wash buffer. Fractions were collected (monitored at 280 nm using UV detector). Bound a-chain was eluted with 8M urea in 50 mM Na-acetate, 500 mM NaCl pH=5.3.
[0282]The purified b-chain was precipitated with addition of 60% TCA to a final concentration of 20%. After a 30 minute incubation at -20° C. for 30 minutes the b-chain preparation was centrifuged at 10,000×g to collect the protein. The TCA supernatant was removed and the precipitate was spun again for 10 seconds at 2000×g to collect TCA drops from the tube wall. Remaining liquid was removed, and the protein pellet was resolubilized in 50 mM Tris, 1% SDS pH=8 at high concentration (for example, 4 mg/ml or higher.) If the pH was less than 7.5-8 it was adjusted with NaOH.
Insulin Quantitation
[0283]The concentration of insulin was determined by measuring the absorbance at 280 nm after zeroing with a solution of 50 mM Tris, 1% SDS pH=8. The insulin-b chain has theoretical absorbance of 0.913 at 1 mg/ml concentration (according to the Swiss-Prot Protein Parameters tool). The concentration can be determined by dividing the actual absorbance of the protein solution accounting for the dilution, by the absorbance of 1 mg/ml solution. C=A×D/0.913, where C is concentration (mg/ml); A is absorbance at 280 nm; and D is dilution.
EXAMPLE 6
Protein Alkylation of Unstained Markers
[0284]Insulin b-Chain
[0285]Alkylation is performed at a protein concentration of 1 mg/ml. 100 μl of 10 mg/ml Insulin-b chain is brought up to a volume of 1 ml in a solution having a final concentration of 50 mM Tris pH=8, 0.5% SDS. 10 μl of 400 mM tributhylphosphine (TBP) in isopropanol was added to the protein sample and the mixture was vortexed for 10-15 seconds. The sample was incubated for 10 minutes at 70° C. and then cooled for 5 minutes at room temperature (or until the temperature dropped to 30° C.). 50 μl of 1M iodoacetamide in ultrapure water was added, and the sample was vortexed for 3-5 seconds and then incubated for 40-60 minutes at room temperature in darkness.
10 kDa BenchMark® Protein Standard
[0286]The 10 kDa BenchMark® protein marker is the recombinantly-expressed truncated E. coli thioredoxin protein that includes amino acids 1-85 from E. coli thioredoxin, a substitution of glutamic acid for valine at amino acid at amino acid position number 86, and histidine residues at positions 87-92 (Trxfusprl10A; see FIG. 3 of U.S. Pat. No. 6,703,484, herein incorporated by reference in its entirety). 100 μl of the 10 kDa BenchMark® stock solution (OD=8.3) was brought up to a volume of 1 ml with a final concentration of 50 mM Tris pH=8 and 0.5% SDS. 10 μl of 400 mM tributhylphosphine (TBP) in isopropanol was added and the protein sample was vortexed for 10-15 seconds and then incubated for 10 minutes at 70° C. The sample was allowed to cool down for 5 minutes at room temperature (or until the temperature dropped to 30° C.) and then 50 μl of 1M iodoacetamide was added and the sample was vortexed for 3-5 seconds, and then incubated for 40-60 minutes at room temperature in darkness.
Lysozyme
[0287]Lysozyme was used as a 15 kDa molecular weight marker. 100 μl of 10 mg/ml lysozyme (Calbiochem, San Diego, Calif., USA) solution in water was brought up to a volume of 1 ml with a final concentration of 50 mM Tris pH=8 and 0.5% SDS. 10 μl of 400 mM tributhylphosphine (TBP) in isopropanol was added and the protein sample was vortexed for 10-15 seconds and then incubated for 10 minutes at 70° C. The sample is allowed to cool down for 5 minutes at room temperature (or until the temperature drops to 30° C.) and then 5.5 μl of 4-vinylpyridine (distilled) is added and the sample is vortexed to solubilize the 4-vinylpyridine, and then incubated for one hour at room temperature in darkness.
20 kDa BenchMark® Protein Standard
[0288]The 20 kDa BenchMark® protein standard includes a truncated thioredoxin fragment fused to two copies of a 5 kDa fragment of the E. coli DEAD-box protein (as disclosed in U.S. Pat. No. 6,703,484, herein incorporated by reference in its entirety). 100 μl of 20 kDa BenchMark® stock solution (OD=8.2) was brought up to a volume of 1 ml with a final concentration of 50 mM Tris pH=8 and 0.5% SDS. 10 μl of 400 mM tributhylphosphine (TBP) in isopropanol was added and the protein sample was vortexed for 10-15 seconds and then incubated for 10 minutes at 70° C. The sample was allowed to cool down for 5 minutes at room temperature (or until the temperature dropped to 30° C.) and then 50 μl of 1M iodoacetamide was added and the sample was vortexed for 3-5 seconds, and then incubated for 40-60 minutes at room temperature in darkness.
30 kDa NL Protein Standard
[0289]The 30 kDa protein that had no lysines (30 kDa NL) was produced from an expression construct as provided in Examples 1 and 3. Alkylation was performed at 0.5 mg/ml protein concentration. 250 μl of 2 mg/ml 30 kDa (NL) stock solution was brought up to 1 ml volume to a final concentration of 50 mM Tris, 0.5% SDS pH=8. 10 μl 400 mM TBP (tributhylphosphine) in isopropanol was added and the protein sample was vortexed for 10-15 seconds. The sample was then incubated for 10 minutes at 70° C. The sample was then cooled for 5 minutes at room temperature (or until the temperature dropped to 30° C.). 50 μl of 1M iodoacetamide was added, and the sample was vortexed for 3-5 seconds and then incubated for 40-60 minutes at room temperature in the dark.
40 kDa NL Protein Standard
[0290]The 40 kDa protein that had no lysines (40 kDa NL) was produced from an expression construct as provided in Examples 1 and 3. Alkylation was performed at 0.5 mg/ml protein concentration. 250 μl of 2 mg/ml 30 kDa (NL) stock solution was brought up to 1 ml volume to a final concentration of 50 mM Tris, 0.5% SDS pH=8. 10 μl 400 mM TBP (tributhylphosphine) in isopropanol was added and the protein sample was vortexed for 10-15 seconds. The sample was then incubated for 10 minutes at 70° C. The sample was then cooled for 5 minutes at room temperature (or until the temperature dropped to 30° C.). 50 μl of 1M iodoacetamide was added, and the sample was vortexed for 3-5 seconds and then incubated for 40-60 minutes at room temperature in the dark.
50 kDa NL Protein Standard
[0291]The 50 kDa protein that had no lysines (50 kDa NL) was produced from an expression construct as provided in Examples 1 and 3. Alkylation was performed at 0.5 mg/ml protein concentration. 250 μl of 2 mg/ml 30 kDa (NL) stock solution was brought up to 1 ml volume to a final concentration of 50 mM Tris, 0.5% SDS pH=8. 10 μl 400 mM TBP (tributhylphosphine) in isopropanol was added and the protein sample was vortexed for 10-15 seconds. The sample was then incubated for 10 minutes at 70° C. The sample was then cooled for 5 minutes at room temperature (or until the temperature dropped to 30° C.). 50 μl of 1M iodoacetamide was added, and the sample was vortexed for 3-5 seconds and then incubated for 40-60 minutes at room temperature in the dark.
60 kDa BenchMark® Protein Standard
[0292]The 60 kDa BenchMark® molecular weight marker protein includes six fused copies of a truncated E. coli thioredoxin protein (see U.S. Pat. No. 6,703,484, herein incorporated by reference in its entirety). 100 μl of 60 kDa BenchMark® stock solution (OD=3.49) was brought up to a volume of 1 ml with a final concentration of 50 mM Tris pH=8 and 0.5% SDS. 10 μl of 400 mM tributhylphosphine (TBP) in isopropanol was added and the protein sample was vortexed for 10-15 seconds and then incubated for 10 minutes at 70° C. The sample was allowed to cool down for 5 minutes at room temperature (or until the temperature dropped to 30° C.) and then 5.5 μl of 4-vinylpyridine (distilled) was added and the sample was vortexed to solubilize the 4-vinylpyridine and then incubated for one hour at room temperature in the dark.
80 kDa BenchMark® Protein Standard
[0293]The 80 kDa BenchMark® molecular weight marker protein includes eight fused copies of a truncated E. coli thioredoxin protein (see U.S. Pat. No. 6,703,484, herein incorporated by reference in its entirety). 100 μl of 60 kDa BenchMark® stock solution (OD=6.36) was brought up to a volume of 1 ml with a final concentration of 50 mM Tris pH=8 and 0.5% SDS. 10 μl of 400 mM tributhylphosphine (TBP) in isopropanol was added and the protein sample was vortexed for 10-15 seconds and then incubated for 10 minutes at 70° C. The sample was allowed to cool down for 5 minutes at room temperature (or until the temperature dropped to 30° C.) and then 5.5 μl of 4-vinylpyridine (distilled) was added and the sample was vortexed to solubilize the 4-vinylpyridine and then incubated for one hour at room temperature in the dark.
110 kDa NL Protein Standard
[0294]The 110 kDa protein that had no lysines (110 kDa NL) was produced from an expression construct as provided in Example 2 and Example 3. Alkylation was performed at 0.5 mg/ml protein concentration. 50 μl 1M Tris pH=8, 25 μl 20% SDS, and 675 μl water were added to 250 μl of a 2 mg/ml stock solution of the 10 kDa (NL) protein. 10 μl 400 mM TBP (tributhylphosphine) in isopropanol was added and the protein sample was vortexed for 10-15 seconds. The sample was then incubated for 10 minutes at 70° C. The sample was then cooled for 5 minutes at room temperature (or until the temperature dropped to 30° C.). 50 μl of 1M iodoacetamide was added, and the sample was vortexed for 3-5 seconds and then incubated for 40-60 minutes at room temperature in the dark.
160 kDa NL Protein Standard
[0295]The 160 kDa protein that had no lysines (160 kDa NL) was produced from an expression construct as provided in Example 2 and Example 3. Alkylation was performed at 0.5 mg/ml protein concentration. 50 μl 1M Tris pH=8, 25 μl 20% SDS, and 675 μl water were added to 250 μl of a 2 mg/ml stock solution of the 160 kDa (NL) protein. 10 μl 400 mM TBP (tributhylphosphine) in isopropanol was added and the protein sample was vortexed for 10-15 seconds. The sample was then incubated for 10 minutes at 70° C. The sample was then cooled for 5 minutes at room temperature (or until the temperature dropped to 30° C.). 50 μl of 1M iodoacetamide was added, and the sample was vortexed for 3-5 seconds and then incubated for 40-60 minutes at room temperature in the dark.
260 kDa Protein Standard
[0296]The 260 kDa protein standard (260 kDa) was produced from an expression construct as provided in Example 2 and Example 3. Alkylation was performed at 0.5 mg/ml protein concentration. 50 μl 1M Tris pH=8, 25 μl 20% SDS, and 675 μl water were added to 250 μl of a 2 mg/ml stock solution of the 260 kDa protein. 10 μl 400 mM TBP (tributhylphosphine) in isopropanol was added and the protein sample was vortexed for 10-15 seconds. The sample was then incubated for 10 minutes at 70° C. The sample was then cooled for 5 minutes at room temperature (or until the temperature dropped to 30° C.). 50 μl of 1M iodoacetamide was added, and the sample was vortexed for 3-5 seconds and then incubated for 40-60 minutes at room temperature in the dark.
Purification of the Alkylated Proteins
[0297]All alkylated proteins were purified on Bio-Gel P-6 gel filtration columns equilibrated with 0.1% SDS in 50 mM Tris pH=8. The protein elution was monitored at 280 nm with a UV detector. The sample volume was 10% or less of the volume of the column.
EXAMPLE 7
Synthesis of Red Dye #1 (8-anilino-1-naphthalenesulfonic acid-aminophenyl vinyl sulfone; 8-ANS-APVS)
[0298]The synthesis of 8-anilino-1-naphthalenesulfonic acid-aminophenyl vinyl sulfone (8-ANS-APVS) involves the use of a diazonium salt which is prone to rapid decomposition and can be hazardous. The diazonium salt should not be allowed to dry out. The synthesis scheme is depicted in FIG. 12.
[0299]4-aminophenyl-2-sulfonatoethyl sulfone (2.81 grams) was placed in a 200 mL round bottom flask equipped with a stir bar. 50 mL of water was added to the flask, followed by 10 mL of concentrated HCl. The mixture was stirred thoroughly and then cooled to 0° C. in an ice water bath. In a separate 50 mL flask, 0.69 g of sodium nitrite was mixed in 20 mL of water until it was completely dissolved. This mixture was added to an addition funnel and placed on top of the flask containing the 4-aminophenyl-2-sulfonatoethyl sulfone. The sodium nitrite solution was added dropwise to the mixture and the solid in the flask began to dissolve with a yellowish/green color developing in the solution. After the addition of sodium nitrite was complete the ice bath was removed and the temperature was allowed to rise to 20° C. The solution became clear as the diazonium salt formed. The solution was then cooled back to 0° C. to precipitate the diazonium salt.
[0300]8-anilino-1-naphthalenesulfonic acid (8-ANS) was prepared by placing the solid in a 250 mL round bottom flask equipped with a stir bar. 30 mL of water was added, followed by 5 mL of 1.0 M sodium carbonate. The mixture was stirred thoroughly until the 8-ANS dissolved. The diazonium salt was transferred to an addition funnel and the diazonium salt solution was added to the solution of 8-ANS dropwise with stirring. A dark color developed immediately. Once the addition was finished the mixture was stirred for at least 2 hours up to overnight.
[0301]The dye was purified by reverse phase chromatography using either methanol or acetonitrile as the eluant. The dye was loaded on the C-18 resin in 50 mM phosphate pH 3.0 (the pH of the aqueous dye solution was increased before loading onto the column to avoid breaking the silane bonds of silica-based C-18 sorbents). The resin-bound dye was then washed to remove most of the acid from the coupling step. At low pH the dye is a purple color and the fractions collected were in some cases checked by HPLC to assess purity. The combined fractions were reduced in vacuo by rotary evaporation at reduced pressure. The yield was calculated by standard methods. The dried dye vinyl sulfone precursor was dissolved in 50 mL of water and transferred to a 100-200 mL round bottom flask equipped with a stir bar. While stirring the solution 5 mL of the 1.0 M sodium carbonate solution was added. The pH was maintained at 10.0±0.2 using a calibrated pH meter. This solution was stirred for 1 hour and then adjusted to pH 7 using 1 N HCl. The dye was purified using a reverse phase column. The reactive dye was loaded directly onto the column after adjusting the pH to 7. The column was washed thoroughly with water after the dye was loaded. The dye was eluted in acetonitrile and the colored fractions were collected. The dye fractions were combined and the solvent was removed in vacuo using a rotary evaporator. The solid dye was weighted and the yield was calculated. A negative ion mode mass spectrum was obtained to be sure that a parent peak was seen at a mass to charge ratio of 492.
EXAMPLE 8
Activation of Orange 16 Dye
[0302]The starting material, Reactive Orange 16 (also called Remazol Brilliant Orange 3R), was obtained from Sigma-Aldrich Chemical Company. It was converted to the vinyl sulfone in order to react with the sulfhydryls of proteins for generating dyed marker proteins. The reaction scheme for generating the vinyl sulfone form of the dye is depicted in FIG. 13.
[0303]A 100 mL round bottom flask was equipped with the appropriate sized egg-shaped stir bar. The flask was charged with Reactive Orange 16 which was dissolved by the required volume of water. With the solution is stirring, sodium hydroxide was added dropwise to the stirred the solution until the pH is 10.0±0.1. The reaction was allowed to stir for 2 hours and while the pH was monitored. 100 μl of 1M sodium carbonate was added to keep the pH at 10.0. After two hours the pH was adjusted back to neutrality using 1 M HCl.
[0304]The was purified by C18 column chromatography. The product was loaded onto a Waters bondapak resin column in 50 mM phosphate pH 4. Once the product was loaded onto the column the column was washed with 3 column volumes of water and then the product was eluted using 50% HPLC grade methanol in water. The fractions were combined and the dark fractions were concentrated in vacuo on a rotary evaporator. The product was scraped from the flask and placed in a tared amber bottle/vial to obtain the weight of product. The bottle was purged with argon and labeled with the following name to distinguish it from the starting material: "Reactive Orange 16 Vinyl Sulfone".
EXAMPLE 9
Labeling of Standard Proteins with Dyes
[0305]The labeling of all no-lysine (NL) proteins (the 30 kDa, 40 kDa, 50 kDa, 110 kDa, and 160 kDa NL proteins) and the 260 kDa protein was performed at 0.5 mg/ml final concentration. The amount of protein and water added to the reactions was adjusted depending on the starting protein concentration. Insulin and lysozyme were labeled at the concentrations described in the corresponding protocols. BenchMark® protein standards are described in U.S. Pat. No. 6,704,484, herein incorporated by reference in its entirety.) The BenchMark® protein standard stock solutions were labeled at constant concentration (the ODs specified in the protocols).
Insulin
[0306]50 μl 1M Tris pH=8, 25 ul 20% SDS, and 875 μl ultrapure water were added to 50 μl of 20 mg/ml Insulin b-chain protein. 2.5 μl 400 mM TBP was added and the protein sample was incubated for 20 minutes at 70° C. The sample was then cooled for 5 minutes at room temperature or until the temperature was below 50° C. 100 μl 10 mg/ml Uniblue A in water was then added to the peptide sample and the sample was incubated for 3 hours at 50° C.
10 kDa BenchMark® Standard
[0307]The BenchMark® 10 kDa protein standard (Invitrogen Corp., Carlsbad, Calif.; U.S. Pat. No. 6,703,484) was labeled for use as the 10 kDa standard of the pre-labeled marker set. 50 μl 1M Tris pH=8, 25 ul 20% SDS, and 825 μl ultrapure water were added to 100 μl of an 8.3 OD solution of 10 kDa BenchMark® protein standard stock solution. 2.5 μl 400 mM TBP was added and the protein sample was incubated for 20 minutes at 70° C. The sample was then cooled for 5 minutes at room temperature or until the temperature was below 30° C. 50 μl 10 mg/ml 8-ANS-APVS in DMF was added to the protein sample and the sample was incubated for 3 hours at room temperature.
Lysozyme
[0308]50 μl 1M Tris pH=8, 25 ul 20% SDS, and 825 μl ultrapure water were added to 100 μl 10 mg/ml lysozyme solution in water. 2.5 μl 400 mM TBP was added and the protein sample was incubated for 20 minutes at 70° C. The sample was then cooled for 5 minutes at room temperature or until the temperature was below 30° C. 50 μl of 10 mg/ml Uniblue in DMF was added to the protein sample and the sample was incubated overnight at room temperature.
20 kDa BenchMark® Protein Standard
[0309]The BenchMark® 20 kDa protein standard, a 19.891 kDa protein having a truncated thioredoxin linked to two copies of a 5 kDa fragment of the Dead-box protein, (Invitrogen Corp., Carlsbad, Calif.; U.S. Pat. No. 6,703,484) was labeled for use as the 20 kDa standard of the pre-labeled marker set. 50 μl 1M Tris pH=8, 25 ul 20% SDS, and 825 μl ultrapure water were added to 100 μl of an 8.2 OD solution of 20 kDa BenchMark® protein standard stock solution. 2.5 μl 400 mM TBP was added and the protein sample was incubated for 20 minutes at 70° C. The sample was then cooled for 5 minutes at room temperature or until the temperature was below 30° C. 12.5 μl of 20 mg/ml Bodipy 530/550 iodoacetamide in DMF was added to the protein sample and the sample was incubated for 3 hours at room temperature.
30 kDa NL Protein Standard
[0310]50 μl 1M Tris pH=8, 25 ul 20% SDS, and 800 μl ultrapure water were added to 125 μl of a 5 mg/ml solution of the 30 kDa NL standard protein. 2.5 μl 400 mM TBP was added and the protein sample was incubated for 20 minutes at 70° C. The sample was then cooled for 5 minutes at room temperature or until the temperature was below 30° C. 100 μl of 10 mg/ml Uniblue A in water was added to the protein sample and the sample was incubated overnight (14-18 hours) at room temperature.
40 kDa NL Protein Standard
[0311]50 μl 1M Tris pH=8, 25 ul 20% SDS, and 725 μl ultrapure water were added to 200 μl of a 2.5 mg/ml solution of the 40 kDa (NL) standard protein. 2.5 μl 400 mM TBP was added and the protein sample was incubated for 20 minutes at 70° C. The sample was then cooled for 5 minutes at room temperature or until the temperature was below 30° C. 50 μl of 40 mg/ml activated Orange 16 in water was added to the protein sample and the sample was incubated for 3 hours at room temperature.
50 kDa NL Protein Standard
[0312]50 μl 1M Tris pH=8, 25 ul 20% SDS, and 800 μl ultrapure water were added to 125 μl of a 5 mg/ml solution of the 50 kDa (NL) standard protein. 2.5 μl 400 mM TBP was added and the protein sample was incubated for 20 minutes at 70° C. The sample was then cooled for 5 minutes at room temperature or until the temperature was below 30° C. 100 μl of 10 mg/ml Uniblue A in water was added to the protein sample and the sample was incubated overnight (14-18 hours) at room temperature.
60 kDa BenchMark® Protein Standard
[0313]The BenchMark® 60 kDa protein standard (Invitrogen Corp., Carlsbad, Calif.; U.S. Pat. No. 6,703,484) was labeled for use as the 60 kDa standard of the pre-labeled marker set. 50 μl 1M Tris pH=8, 25 ul 20% SDS, and 665 μl ultrapure water were added to 260 μl of a 3.49 OD solution of 60 kDa BenchMark® standard protein stock solution. 2.5 μl 400 mM TBP was added and the protein sample was incubated for 20 minutes at 70° C. The sample was then cooled for 5 minutes at room temperature or until the temperature was below 30° C. 50 μl of 10 mg/ml 8-ANS-APVS in DMF was added to the protein sample and the sample was incubated for 6 hours at room temperature.
80 kDa BenchMark® Protein Standard
[0314]The BenchMark® 80 kDa protein standard (Invitrogen Corp., Carlsbad, Calif.; U.S. Pat. No. 6,703,484) was labeled for use as the 80 kDa standard of the pre-labeled marker set. 50 μl 1M Tris pH=8, 25 ul 20% SDS, and 825 μl ultrapure water were added to 100 μl of a 6.36 OD solution of 80 kDa BenchMark® protein standard stock solution. 2.5 μl 400 mM TBP was added and the protein sample was incubated for 20 minutes at 70° C. The sample was then cooled for 5 minutes at room temperature or until the temperature was below 30° C. 12.5 of 20 mg/ml Bodipy 530/550 iodoacetamide in DMF was added to the protein sample and the sample was incubated for 2.5 hours at room temperature.
110 kDa NL Protein Standard
[0315]50 μl 1M Tris pH=8, 25 ul 20% SDS, and 800 μl ultrapure water were added to 125 μl of a 4 mg/ml solution of the 110 kDa (NL) standard protein. 2.5 μl 400 mM TBP was added and the protein sample was incubated for 20 minutes at 70° C. The sample was then cooled for 5 minutes at room temperature or until the temperature was below 30° C. 100 μl of 10 mg/ml Uniblue A in water was added to the protein sample and the sample was incubated overnight (14-18 hours) at room temperature.
160 kDa NL Protein Standard
[0316]50 μl 1M Tris pH=8, 25 ul 20% SDS, and 800 μl ultrapure water were added to 125 μl of a 4 mg/ml solution of the 160 kDa (NL) standard protein. 2.5 μl 400 mM TBP was added and the protein sample was incubated for 20 minutes at 70° C. The sample was then cooled for 5 minutes at room temperature or until the temperature was below 30° C. 100 μl of 20 mg/ml Orange 16 in DMF was added to the protein sample and the sample was incubated for 3 hours at 50° C.
260 kDa Protein Standard
[0317]50 μl 1M Tris pH=8, 25 ul 20% SDS, and 725 μl ultrapure water were added to 200 μl of a 2.5 mg/ml solution of the 260 kDa standard protein. 2.5 μl 400 mM TBP was added and the protein sample was incubated for 20 minutes at 70° C. The sample was then cooled for 5 minutes at room temperature or until the temperature was below 30° C. 25 of 20 mg/ml Bodipy 530/550 Iodoacetamide in DMF was added to the protein sample and the sample was incubated for 5-6 hours at room temperature.
Purification of Labeled Proteins
[0318]All of the standard proteins except lysozyme were purified on gel filtration LC column packed with Toyopearl HW-40c resin. The volume of the column was at least 15 times the volume of the sample for the proteins labeled with Uniblue A, Orange 16 and Bodipy 530/550 dyes. The volume of the column was at least 20 times the volume of the sample for proteins labeled with the Red (8-ANS-APVS) dye.
[0319]For purification of lysozyme labeled with Uniblue-A, Bio-Gel P-6 column equilibrated with 8M urea was used. The column had a volume of at least 20 times the sample volume. The sample was loaded on the column and the dye was separated from the protein conjugate. Two dye peaks were seen. After the sample is collected the urea was exchanged to Tris/SDS by loading the sample onto a Bio-Gel P-6 column equilibrated with 50 mM Tris, 0.5% SDS. (The column volume was at least ten times the sample volume.)
Capping of Labeled Proteins
[0320]A capping step was performed to neutralize any unreacted cysteine residues on the standard proteins to prevent the proteins from forming intra and inter disulfide bridges which could lead to changes in electrophoretic migration and reduce band sharpness on gels. Labeled proteins were denatured and reduced with the addition of 25 μl of 20% SDS and 10 μl 400 mM TBP per 1 ml of protein conjugate with an incubation of 30 minutes at room temperature. Then 50 μl of 1M iodoacetamide was added per 1 ml of protein conjugate and the sample was incubated for 1 hour at room temperature. In the case of lysozyme SDS was not added prior to the reaction since the SDS concentration of the lysozyme standard solution was already at 0.5%. 10 μl 400 mM TBP were added per 1 ml of protein conjugate and sample incubated for 30 minutes at room temperature. Then 50 μl of 1M iodoacetamide was added per 1 ml of protein conjugate and the sample was incubated for 1 hour at room temperature.
[0321]The unreacted reducing and alkylation reagents were removed from the labeled, alkylated proteins by gel filtration on Bio-Gel P-6 columns equilibrated with 0.1% SDS in 50 mM Tris pH=8.
Sephacryl Purification of the Labeled Proteins
[0322]In some cases a second purification of a standard protein was performed on Sephacryl column. Sephacryl 200-HR was used for proteins of 10 kDa to 30 kDa and Sephacryl 400-HR was used for proteins with molecular weight of 40 kDa to 260 kDa. The columns were washed with 50 mM Tris, 0.1% SDS and then the sample was loaded.
[0323]The column had a volume of at least 30 times the sample volume and length to internal diameter ratio of at least 20 (for example 100 cm×5 cm ID column can be used for the purification 100 ml sample. Fractions of 10 ml were collected and aliquots were run on a gel, and the purified protein fractions were pooled together.
Concentration
[0324]Standard proteins were concentrated on Vivaspin MWCO filters with suitable pore size: 100 kDa MWCO filter for 260 kDa, 160 kDa and 110 kDa standard proteins; 50 kDa MWCO filter for 80 kDa, 60 kDa and 50 kDa standard proteins; 30 kDa MWCO filter for 40 kDa and 30 kDa standard proteins; 10 kDa MWCO filter for 20 kDa, lysozyme, and 10 kDa standard proteins; 3 kDa MWCO filter for insulin b-chain.
EXAMPLE 10
Electrophoretic Migration
[0325]Each of the prestained proteins was loaded side by side with the corresponding unlabeled protein marker on gels. The samples were analyzed for migration on 8 cm×8 cm 4-12% BisTris/MES gels, 4-12% BisTris/MOPS gels, and 4-20% Tris Glycine gels. The gels were run at 200 V until the dye front reached the bottom of the gel (6.8 cm from the bottom of the sample wells). After electrophoresis the gel was stained with SimplyBlue® Safe Stain Coomassie G-250® protein stain (Invitrogen Corp., Carlsbad, Calif.) according to the microwave protocol. The gels were destained for several hours to overnight with deionized water. The migration of the labeled proteins was measured on Alpha Imager 3000 imaging system. Pictures of the gels were taken with the Alpha Imager and the migration of the labeled proteins were analyzed relative to the same protein standard in unlabeled form. Point-to-point calibration was used to increase the accuracy of the measurement in calculating the molecular weights of the proteins based on their migration distances, in which a standard curve was generated by plotting the log of molecular weight versus migration distance for the two protein markers migrating closest to the protein whose molecular weight was being calculated (one that migrated a shorter distance than the protein standard whose weight was being calculated and the other that migrated a longer distance than the protein standard whose weight was being calculated.) The pre-labeled protein standards were observed to migrate substantially the same as their unlabeled counterparts when the molecular weights were calculated from the point-to-point calibration were within 10%. All of the labeled molecular weight marker proteins having molecular weights of 10 kDa or greater migrated within 4.5% of the migration of their unlabeled counterparts.
EXAMPLE 11
Sharp Pre-Stained Standard Protein Blend Preparation
[0326]Twelve labeled proteins (insulin b-chain, 10 kDa BenchMark® protein Standard, 20 kDa BenchMark® protein Standard, 30 kDa NL protein Standard, 40 kDa NL protein Standard, 50 kDa NL protein Standard, 60 kDa BenchMark® protein Standard, 80 kDa BenchMark® protein Standard, 110 kDa NL protein Standard, 160 kDa NL protein Standard, and 260 kDa protein Standard) were blended to make a molecular weight standard set in which the molecular weights of the protein standards ranged from less than 3.5 kDa to greater than 250 kDa. The molecular weight standard set included proteins labeled with four different visually distinguishable dyes. The proteins were blended for consistent batch-to-batch intensity by comparing the intensity of the bands from each new preparation of labeled standard to a prior batch of standard to provide standards with no more than 20% variation in the band intensities from batch to batch. An appropriate amount of each protein standard was added to the blend and ultra pure water was added to 50% of the target final volume. Then 50% of the target final volume of 2× Sample Buffer (130 mM Tris pH=6.5, 4% SDS, 60% Glycerol, 0.01% Coomassie G 250) was added to the marker blend preparation.
[0327]An unlabeled standard set comprising the same proteins as the pre-labeled set was also formulated. The unlabeled standard set was formulated such that the 20 kDa and 80 kDa standard protein bands were more intense than the other protein bands when viewed on an electrophoresis gel, so that the user can orient the proteins readily by observation of the intense 20 kDa and 80 kD bands.
TABLE-US-00005 TABLE 4 Sharp Pre-stained Standard Proteins Molecular Actual Weight to Molecular Conjugated Visible nearest 1 Weight Protein Standard Dye Color kDa (kDa) Insulin B-chain Uniblue A Blue 3 3.4 (seq) 3.5 kDa protein Standard BenchMark ® ANS-APVS Red 10 10.170 10 kDa protein (Red dye #1) 10.172 (ms) Standard Lysozyme Uniblue A Blue 14 15 kDa protein Standard BenchMark ® Bodipy Pink 20 19.892 (seq) 20 kDa protein 530/550 19.906 (ms) Standard No-lysine Uniblue A Blue 30 30.012 30 kDa protein 29.979 (ms) Standard No-lysine Orange 16 Orange 40 40.123 (ms) 40 kDa protein Standard No-lysine Uniblue A Blue 50 50.253 (seq) 50 kDa protein 50.044 (ms) Standard BenchMark ® ANS-APVS Red 60 59.738 (ms) 60 kDa protein (Red dye #1) Standard BenchMark ® Bodipy Pink 80 79.785 (ms) 80 kDa protein 530/550 Standard No-lysine Uniblue A Blue 110 109.798 (ms) 110 kDa protein Standard No-lysine Orange 16 Orange 159 158.843 (ms) 160 kDa protein Standard 260 kDa protein Bodipy Pink 262 262.379 (ms) Standard 530/550
EXAMPLE 12
Electophoresis of a Pre-Labeled Protein Standard Set
[0328]The pre-labeled marker set of Example 11 (10 microliters) was electrophoresed alongside the same set of proteins in unlabeled form (5 microliters) in a 4-12% Bis-Tris (NuPAGE® Novex®) acrylamide gel run with 1×MES buffer. FIG. 14 shows that the pre-labeled protein standard set that includes five proteins labeled on cysteine and lacking lysine has twelve bands that produce sharp bands that migrate substantially the same as their unlabeled counterparts. The pre-labeled marker set of Example 11 was also electrophoresed on a 4-12% Bis-Tris (NuPAGE® Novex®) acrylamide gel run with 1×MES buffer, a 4-12% Bis-Tris (NuPAGE® Novex®) acrylamide gel run with 1×MOPS buffer, and a 4-20% Tris-glycine (Novex®) gel (FIG. 15) alongside other commercially available markers (1, Precision Plus Blue (Bio-Rad); 2, Precision Plus Dual (Bio-Rad); 3, Precision Plus Kaleidoscope (Bio-Rad); 4, Sharp Pre-stained Standard (Invitrogen); 5--Rainbow (GE); 6--BenchMark® prestain (Invitrogen); 7--MultiMark (Invitrogen); 8--SeeBlue+2 (Invitrogen). All gels were 8×8 cm "mini" gels from Invitrogen, Carlsbad, Calif., and electrophoresis conditions were those provided by the manufacturer.
TABLE-US-00006 TABLE 5 Migration of Pre-labeled Standard Set on BisTris Gels Band 4-12% Gel/MES 4-12% Gel/MOPS 260 kDa 0% -2.1% 160 kDa -4.1% -4.5% 110 kDa -3.7% -2.7% 80 kDa +1.5% +3.7% 60 kDa -0.4% +0.6% 50 kDa -3.4% -3.3% 40 kDa +1% +0.3% 30 kDa -0.5% -1.6% 20 kDa +2.2% +0.5% 15 kDa 0% -3.5% 10 kDa +1.2% -0.1%* 3.5 kDa +10%**
TABLE-US-00007 TABLE 6 Migration of Pre-labeled Standard Set on 4-20% Tris glycine gel Bands Percent difference 260 kDa 0% 160 kDa -1.2% 110 kDa -1.8% 80 kDa +4.3% 60 kDa -1% 50 kDa +0.3% 40 kDa +3.6 30 kDa -0.6% 20 kDa 0% 15 kDa +1.3% 10 kDa 0%
EXAMPLE 13
Calculation of Band Widths of Electophoresed Proteins of a Pre-Labeled Protein Standard Set
[0329]10 ul Sharp Pre-stained Protein Standard formulation of Example 11 was run on a 4-12% acrylamide gradient Bis-Tris NuPAGE® gel run with 1×MES running buffer (Invitrogen, Carlsbad, Calif.). After electrophoresis the gel was placed on a transparency having a copy of a measuring scale (FIG. 16A). The gel was then scanned at 300/300 dpi and saved as gray scale `.BMP` image.
[0330]The resolution of the gel was later decreased across the width (to make it compatible with Gelo.exe). The resulting gel image was loaded in Gelo.exe, a software program designed to measure dimensions of an image, and a trace was extracted of image intensity down the length of the gel.
[0331]The extracted trace was loaded in Peakman.exe. The baseline was adjusted and peaks were selected. The Peakman.exe program measured the width of the bands where the intensity of the image was 50% or more of the maximum intensity peak height for (FIG. 16B). The data was loaded in Excel and the number of image units per 1 mm was calculated by dividing the length of the gel by the total number of image units for this length: Running length of the gel=68 mm; Length in image units=850-44=806; Number of image units per 1 mm=806/68=11.85. The width of each peak at half height was therefore divided by 11.85 to obtain the width in millimeters.
[0332]The widths of the bands produced by the electrophoreses protein standard (peaks 2-13, corresponding to pre-stained protein bands on the gel), are provided in Table 7. The width of bands visible to the naked eye from proteins having a molecular weight of greater than 3.5 kDa range in width from 0.59 mm to 1.16 mm, a difference of just under 2-fold. The width of bands visible to the naked eye from proteins having a molecular weight of at least 10 kDa to 110 kDa or less range in width from 0.73 mm to 1.16 mm, a difference of less than 1.5-fold. The width of bands visible to the naked eye from proteins having a molecular weight of at least 20 kDa to less than 100 kDa range in width from 0.99 mm to 1.16 mm, a difference of less than 20%. The markers include 6 proteins having a molecular weight of at least 20 kDa to less than 100 kDa, in which the width of the bands visible to the naked eye of the electrophoresed proteins differ by less than 20%.
TABLE-US-00008 TABLE 7 Band Widths of Sharp Pre-stained Standard Proteins Half- Half height Peak height- Width Band Height Width (mm) 260 kDa 0.117 8.496 0.72 160 kDa 0.070 6.968 0.59 110 kDa 0.048 8.623 0.73 80 kDa 0.107 9.506 0.80 60 kDa 0.075 11.85 1.00 50 kDa 0.056 11.439 0.97 40 kDa 0.090 11.627 0.98 30 kDa 0.050 11.652 0.98 20 kDa 0.126 11.789 0.99 15 kDa 0.058 13.709 1.16 10 kDa 0.082 12.518 1.06 3.5 kDa 0.056 14.581 1.23
[0333]The intensity of the bands, as seen by the Peak Height column, varies by no more than 2.5-fold among the proteins of the set.
[0334]Although various embodiments of the invention have been described and provided in the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. In particular, elements and features of embodiments described herein can be combined with elements and features of other embodiments described herein or known in the art to produce further embodiments within the scope of the invention. Headings have been provided solely for the convenience of the reader, and do not limit the scope of the invention.
[0335]All publications, patents and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains, and are herein incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference.
Sequence CWU
1
411109PRTArtificialEscherchia coli 1Met Ser Asp Lys Ile Ile His Leu Thr
Asp Asp Ser Phe Asp Thr Asp1 5 10
15Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu
Trp20 25 30Cys Gly Pro Cys Lys Met Ile
Ala Pro Ile Leu Asp Glu Ile Ala Asp35 40
45Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn50
55 60Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg
Gly Ile Pro Thr Leu Leu65 70 75
80Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu
Ser85 90 95Lys Gly Gln Leu Lys Glu Phe
Leu Asp Ala Asn Leu Ala100 1052105PRTArtificialHomo
sapiens 2Met Val Lys Gln Ile Glu Ser Lys Thr Ala Phe Gln Glu Ala Leu Asp1
5 10 15Ala Ala Gly Asp
Lys Leu Val Val Val Asp Phe Ser Ala Thr Trp Cys20 25
30Gly Pro Cys Lys Met Ile Lys Pro Phe Phe His Ser Leu Ser
Glu Lys35 40 45Tyr Ser Asn Val Ile Phe
Leu Glu Val Asp Val Asp Asp Cys Gln Asp50 55
60Val Ala Ser Glu Cys Glu Val Lys Cys Met Pro Thr Phe Gln Phe Phe65
70 75 80Lys Lys Gly Gln
Lys Val Gly Glu Phe Ser Gly Ala Asn Lys Glu Lys85 90
95Leu Glu Ala Thr Ile Asn Glu Leu Val100
105385PRTArtificialEscherchia coli 3Met Gln Thr Val Ile Phe Gly Arg Ser
Gly Cys Pro Tyr Cys Val Arg1 5 10
15Ala Lys Asp Leu Ala Glu Lys Leu Ser Asn Glu Arg Asp Asp Phe
Gln20 25 30Tyr Gln Tyr Val Asp Ile Arg
Ala Glu Gly Ile Thr Lys Glu Asp Leu35 40
45Gln Gln Lys Ala Gly Lys Pro Val Glu Thr Val Pro Gln Ile Phe Val50
55 60Asp Gln Gln His Ile Gly Gly Tyr Thr Asp
Phe Ala Ala Trp Val Lys65 70 75
80Glu Asn Leu Asp Ala854215PRTArtificialEscherchia coli 4Met Lys
Leu Tyr Ile Tyr Asp His Cys Pro Tyr Cys Leu Lys Ala Arg1 5
10 15Met Ile Phe Gly Leu Lys Asn Ile
Pro Val Glu Leu His Val Leu Leu20 25
30Asn Asp Asp Ala Glu Thr Pro Thr Arg Met Val Gly Gln Lys Gln Val35
40 45Pro Ile Leu Gln Lys Asp Asp Ser Arg Tyr
Met Pro Glu Ser Met Asp50 55 60Ile Val
His Tyr Val Asp Lys Leu Asp Gly Lys Pro Leu Leu Thr Gly65
70 75 80Lys Arg Ser Pro Ala Ile Glu
Glu Trp Leu Arg Lys Val Asn Gly Tyr85 90
95Ala Asn Lys Leu Leu Leu Pro Arg Phe Ala Lys Ser Ala Phe Asp Glu100
105 110Phe Ser Thr Pro Ala Ala Arg Lys Tyr
Phe Val Asp Lys Lys Glu Ala115 120 125Ser
Ala Gly Asn Phe Ala Asp Leu Leu Ala His Ser Asp Gly Leu Ile130
135 140Lys Asn Ile Ser Asp Asp Leu Arg Ala Leu Asp
Lys Leu Ile Val Lys145 150 155
160Pro Asn Ala Val Asn Gly Glu Leu Ser Glu Asp Asp Ile Gln Leu
Phe165 170 175Pro Leu Leu Arg Asn Leu Thr
Leu Val Ala Gly Ile Asn Trp Pro Ser180 185
190Arg Val Ala Asp Tyr Arg Asp Asn Met Ala Lys Gln Thr Gln Ile Asn195
200 205Leu Leu Ser Ser Met Ala Ile210
2155450PRTArtificialEscherchia coli 5Met Thr Lys His Tyr Asp Tyr
Ile Ala Ile Gly Gly Gly Ser Gly Gly1 5 10
15Ile Ala Ser Ile Asn Arg Ala Ala Met Tyr Gly Gln Lys
Cys Ala Leu20 25 30Ile Glu Ala Lys Glu
Leu Gly Gly Thr Cys Val Asn Val Gly Cys Val35 40
45Pro Lys Lys Val Met Trp His Ala Ala Gln Ile Arg Glu Ala Ile
His50 55 60Met Tyr Gly Pro Asp Tyr Gly
Phe Asp Thr Thr Ile Asn Lys Phe Asn65 70
75 80Trp Glu Thr Leu Ile Ala Ser Arg Thr Ala Tyr Ile
Asp Arg Ile His85 90 95Thr Ser Tyr Glu
Asn Val Leu Gly Lys Asn Asn Val Asp Val Ile Lys100 105
110Gly Phe Ala Arg Phe Val Asp Ala Lys Thr Leu Glu Val Asn
Gly Glu115 120 125Thr Ile Thr Ala Asp His
Ile Leu Ile Ala Thr Gly Gly Arg Pro Ser130 135
140His Pro Asp Ile Pro Gly Val Glu Tyr Gly Ile Asp Ser Asp Gly
Phe145 150 155 160Phe Ala
Leu Pro Ala Leu Pro Glu Arg Val Ala Val Val Gly Ala Gly165
170 175Tyr Ile Ala Val Glu Leu Ala Gly Val Ile Asn Gly
Leu Gly Ala Lys180 185 190Thr His Leu Phe
Val Arg Lys His Ala Pro Leu Arg Ser Phe Asp Pro195 200
205Met Ile Ser Glu Thr Leu Val Glu Val Met Asn Ala Glu Gly
Pro Gln210 215 220Leu His Thr Asn Ala Ile
Pro Lys Ala Val Val Lys Asn Thr Asp Gly225 230
235 240Ser Leu Thr Leu Glu Leu Glu Asp Gly Arg Ser
Glu Thr Val Asp Cys245 250 255Leu Ile Trp
Ala Ile Gly Arg Glu Pro Ala Asn Asp Asn Ile Asn Leu260
265 270Glu Ala Ala Gly Val Lys Thr Asn Glu Lys Gly Tyr
Ile Val Val Asp275 280 285Lys Tyr Gln Asn
Thr Asn Ile Glu Gly Ile Tyr Ala Val Gly Asp Asn290 295
300Thr Gly Ala Val Glu Leu Thr Pro Val Ala Val Ala Ala Gly
Arg Arg305 310 315 320Leu
Ser Glu Arg Leu Phe Asn Asn Lys Pro Asp Glu His Leu Asp Tyr325
330 335Ser Asn Ile Pro Thr Val Val Phe Ser His Pro
Pro Ile Gly Thr Val340 345 350Gly Leu Thr
Glu Pro Gln Ala Arg Glu Gln Tyr Gly Asp Asp Gln Val355
360 365Lys Val Tyr Lys Ser Ser Phe Thr Ala Met Tyr Thr
Ala Val Thr Thr370 375 380His Arg Gln Pro
Cys Arg Met Lys Leu Val Cys Val Gly Ser Glu Glu385 390
395 400Lys Ile Val Gly Ile His Gly Ile Gly
Phe Gly Met Asp Glu Met Leu405 410 415Gln
Gly Phe Ala Val Ala Leu Lys Met Gly Ala Thr Lys Lys Asp Phe420
425 430Asp Asn Thr Val Ala Ile His Pro Thr Ala Ala
Glu Glu Phe Val Thr435 440 445Met
Arg4506522PRTArtificialHomo sapiens 6Met Ala Leu Leu Pro Arg Ala Leu Ser
Ala Gly Ala Gly Pro Ser Trp1 5 10
15Arg Arg Ala Ala Arg Ala Phe Arg Gly Phe Leu Leu Leu Leu Pro
Glu20 25 30Pro Ala Ala Leu Thr Arg Ala
Leu Ser Arg Ala Met Ala Cys Arg Gln35 40
45Glu Pro Gln Pro Gln Gly Pro Pro Pro Ala Ala Gly Ala Val Ala Ser50
55 60Tyr Asp Tyr Leu Val Ile Gly Gly Gly Ser
Gly Gly Leu Ala Ser Ala65 70 75
80Arg Arg Ala Ala Glu Leu Gly Ala Arg Ala Ala Val Val Glu Ser
His85 90 95Lys Leu Gly Gly Thr Cys Val
Asn Val Gly Cys Val Pro Lys Lys Val100 105
110Met Trp Asn Thr Ala Val His Ser Glu Phe Met His Asp His Ala Asp115
120 125Tyr Gly Phe Pro Ser Cys Glu Gly Lys
Phe Asn Trp Arg Val Ile Lys130 135 140Glu
Lys Arg Asp Ala Tyr Val Ser Arg Leu Asn Ala Ile Tyr Gln Asn145
150 155 160Asn Leu Thr Lys Ser His
Ile Glu Ile Ile Arg Gly His Ala Ala Phe165 170
175Thr Ser Asp Pro Lys Pro Thr Ile Glu Val Ser Gly Lys Lys Tyr
Thr180 185 190Ala Pro His Ile Leu Ile Ala
Thr Gly Gly Met Pro Ser Thr Pro His195 200
205Glu Ser Gln Ile Pro Gly Ala Ser Leu Gly Ile Thr Ser Asp Gly Phe210
215 220Phe Gln Leu Glu Glu Leu Pro Gly Arg
Ser Val Ile Val Gly Ala Gly225 230 235
240Tyr Ile Ala Val Glu Met Ala Gly Ile Leu Ser Ala Leu Gly
Ser Lys245 250 255Thr Ser Leu Met Ile Arg
His Asp Lys Val Leu Arg Ser Phe Asp Ser260 265
270Met Ile Ser Thr Asn Cys Thr Glu Glu Leu Glu Asn Ala Gly Val
Glu275 280 285Val Leu Lys Phe Ser Gln Val
Lys Glu Val Lys Lys Thr Leu Ser Gly290 295
300Leu Glu Val Ser Met Val Thr Ala Val Pro Gly Arg Leu Pro Val Met305
310 315 320Thr Met Ile Pro
Asp Val Asp Cys Leu Leu Trp Ala Ile Gly Arg Val325 330
335Pro Asn Thr Lys Asp Leu Ser Leu Asn Lys Leu Gly Ile Gln
Thr Asp340 345 350Asp Lys Gly His Ile Ile
Val Asp Glu Phe Gln Asn Thr Asn Val Lys355 360
365Gly Ile Tyr Ala Val Gly Asp Val Cys Gly Lys Ala Leu Leu Thr
Pro370 375 380Val Ala Ile Ala Ala Gly Arg
Lys Leu Ala His Arg Leu Phe Glu Tyr385 390
395 400Lys Glu Asp Ser Lys Leu Asp Tyr Asn Asn Ile Pro
Thr Val Val Phe405 410 415Ser His Pro Pro
Ile Gly Thr Val Gly Leu Thr Glu Asp Glu Ala Ile420 425
430His Lys Tyr Gly Ile Glu Asn Val Lys Thr Tyr Ser Thr Ser
Phe Thr435 440 445Pro Met Tyr His Ala Val
Thr Lys Arg Lys Thr Lys Cys Val Met Lys450 455
460Met Val Cys Ala Asn Lys Glu Glu Lys Val Val Gly Ile His Met
Gln465 470 475 480Gly Leu
Gly Cys Asp Glu Met Leu Gln Gly Phe Ala Val Ala Val Lys485
490 495Met Gly Ala Thr Lys Ala Asp Phe Asp Asn Thr Val
Ala Ile His Pro500 505 510Thr Ser Ser Glu
Glu Leu Val Thr Leu Arg515 5207474PRTArtificialEscherchia
coli 7Met Ser Thr Glu Ile Lys Thr Gln Val Val Val Leu Gly Ala Gly Pro1
5 10 15Ala Gly Tyr Ser Ala
Ala Phe Arg Cys Ala Asp Leu Gly Leu Glu Thr20 25
30Val Ile Val Glu Arg Tyr Asn Thr Leu Gly Gly Val Cys Leu Asn
Val35 40 45Gly Cys Ile Pro Ser Lys Ala
Leu Leu His Val Ala Lys Val Ile Glu50 55
60Glu Ala Lys Ala Leu Ala Glu His Gly Ile Val Phe Gly Glu Pro Lys65
70 75 80Thr Asp Ile Asp Lys
Ile Arg Thr Trp Lys Glu Lys Val Ile Asn Gln85 90
95Leu Thr Gly Gly Leu Ala Gly Met Ala Lys Gly Arg Lys Val Lys
Val100 105 110Val Asn Gly Leu Gly Lys Phe
Thr Gly Ala Asn Thr Leu Glu Val Glu115 120
125Gly Glu Asn Gly Lys Thr Val Ile Asn Phe Asp Asn Ala Ile Ile Ala130
135 140Ala Gly Ser Arg Pro Ile Gln Leu Pro
Phe Ile Pro His Glu Asp Pro145 150 155
160Arg Ile Trp Asp Ser Thr Asp Ala Leu Glu Leu Lys Glu Val
Pro Glu165 170 175Arg Leu Leu Val Met Gly
Gly Gly Ile Ile Gly Leu Glu Met Gly Thr180 185
190Val Tyr His Ala Leu Gly Ser Gln Ile Asp Val Val Glu Met Phe
Asp195 200 205Gln Val Ile Pro Ala Ala Asp
Lys Asp Ile Val Lys Val Phe Thr Lys210 215
220Arg Ile Ser Lys Lys Phe Asn Leu Met Leu Glu Thr Lys Val Thr Ala225
230 235 240Val Glu Ala Lys
Glu Asp Gly Ile Tyr Val Thr Met Glu Gly Lys Lys245 250
255Ala Pro Ala Glu Pro Gln Arg Tyr Asp Ala Val Leu Val Ala
Ile Gly260 265 270Arg Val Pro Asn Gly Lys
Asn Leu Asp Ala Gly Lys Ala Gly Val Glu275 280
285Val Asp Asp Arg Gly Phe Ile Arg Val Asp Lys Gln Leu Arg Thr
Asn290 295 300Val Pro His Ile Phe Ala Ile
Gly Asp Ile Val Gly Gln Pro Met Leu305 310
315 320Ala His Lys Gly Val His Glu Gly His Val Ala Ala
Glu Val Ile Ala325 330 335Gly Lys Lys His
Tyr Phe Asp Pro Lys Val Ile Pro Ser Ile Ala Tyr340 345
350Thr Glu Pro Glu Val Ala Trp Val Gly Leu Thr Glu Lys Glu
Ala Lys355 360 365Glu Lys Gly Ile Ser Tyr
Glu Thr Ala Thr Phe Pro Trp Ala Ala Ser370 375
380Gly Arg Ala Ile Ala Ser Asp Cys Ala Asp Gly Met Thr Lys Leu
Ile385 390 395 400Phe Asp
Lys Glu Ser His Arg Val Ile Gly Gly Ala Ile Val Gly Thr405
410 415Asn Gly Gly Glu Leu Leu Gly Glu Ile Gly Leu Ala
Ile Glu Met Gly420 425 430Cys Asp Ala Glu
Asp Ile Ala Leu Thr Ile His Ala His Pro Thr Leu435 440
445His Glu Ser Val Gly Leu Ala Ala Glu Val Phe Glu Gly Ser
Ile Thr450 455 460Asp Leu Pro Asn Pro Lys
Ala Lys Lys Lys465 4708509PRTArtificialHomo sapiens 8Met
Gln Ser Trp Ser Arg Val Tyr Cys Ser Leu Ala Lys Arg Gly His1
5 10 15Phe Asn Arg Ile Ser His Gly
Leu Gln Gly Leu Ser Ala Val Pro Leu20 25
30Arg Thr Tyr Ala Asp Gln Pro Ile Asp Ala Asp Val Thr Val Ile Gly35
40 45Ser Gly Pro Gly Gly Tyr Val Ala Ala Ile
Lys Ala Ala Gln Leu Gly50 55 60Phe Lys
Thr Val Cys Ile Glu Lys Asn Glu Thr Leu Gly Gly Thr Cys65
70 75 80Leu Asn Val Gly Cys Ile Pro
Ser Lys Ala Leu Leu Asn Asn Ser His85 90
95Tyr Tyr His Met Ala His Gly Lys Asp Phe Ala Ser Arg Gly Ile Glu100
105 110Met Ser Glu Val Arg Leu Asn Leu Asp
Lys Met Met Glu Gln Lys Ser115 120 125Thr
Ala Val Lys Ala Leu Thr Gly Gly Ile Ala His Leu Phe Lys Gln130
135 140Asn Lys Val Val His Val Asn Gly Tyr Gly Lys
Ile Thr Gly Lys Asn145 150 155
160Gln Val Thr Ala Thr Lys Ala Asp Gly Gly Thr Gln Val Ile Asp
Thr165 170 175Lys Asn Ile Leu Ile Ala Thr
Gly Ser Glu Val Thr Pro Phe Pro Gly180 185
190Ile Thr Ile Asp Glu Asp Thr Ile Val Ser Ser Thr Gly Ala Leu Ser195
200 205Leu Lys Lys Val Pro Glu Lys Met Val
Val Ile Gly Ala Gly Val Ile210 215 220Gly
Val Glu Leu Gly Ser Val Trp Gln Arg Leu Gly Ala Asp Val Thr225
230 235 240Ala Val Glu Phe Leu Gly
His Val Gly Gly Val Gly Ile Asp Met Glu245 250
255Ile Ser Lys Asn Phe Gln Arg Ile Leu Gln Lys Gln Gly Phe Lys
Phe260 265 270Lys Leu Asn Thr Lys Val Thr
Gly Ala Thr Lys Lys Ser Asp Gly Lys275 280
285Ile Asp Val Ser Ile Glu Ala Ala Ser Gly Gly Lys Ala Glu Val Ile290
295 300Thr Cys Asp Val Leu Leu Val Cys Ile
Gly Arg Arg Pro Phe Thr Lys305 310 315
320Asn Leu Gly Leu Glu Glu Leu Gly Ile Glu Leu Asp Pro Arg
Gly Arg325 330 335Ile Pro Val Asn Thr Arg
Phe Gln Thr Lys Ile Pro Asn Ile Tyr Ala340 345
350Ile Gly Asp Val Val Ala Gly Pro Met Leu Ala His Lys Ala Glu
Asp355 360 365Glu Gly Ile Ile Cys Val Glu
Gly Met Ala Gly Gly Ala Val His Ile370 375
380Asp Tyr Asn Cys Val Pro Ser Val Ile Tyr Thr His Pro Glu Val Ala385
390 395 400Trp Val Gly Lys
Ser Glu Glu Gln Leu Lys Glu Glu Gly Ile Glu Tyr405 410
415Lys Val Gly Lys Phe Pro Phe Ala Ala Asn Ser Arg Ala Lys
Thr Asn420 425 430Ala Asp Thr Asp Gly Met
Val Lys Ile Leu Gly Gln Lys Ser Thr Asp435 440
445Arg Val Leu Gly Ala His Ile Leu Gly Pro Gly Ala Gly Glu Met
Val450 455 460Asn Glu Ala Ala Leu Ala Leu
Glu Tyr Gly Ala Ser Cys Glu Asp Ile465 470
475 480Ala Arg Val Cys His Ala His Pro Thr Leu Ser Glu
Ala Phe Arg Glu485 490 495Ala Asn Leu Ala
Ala Ser Phe Gly Lys Ser Ile Asn Phe500
5059279DNAArtificialTruncated E. coli thioredoxin ORF with
C-terminal his tag 9atgagcgata aaattattca cctgactgac gacagttttg
acacggatgt actcaaagcg 60gacggggcga tcctcgtcga tttctgggca gagtggtgcg
gtccgtgcaa aatgatcgcc 120ccgattctgg atgaaatcgc tgacgaatat cagggcaaac
tgaccgttgc aaaactgaac 180atcgatcaaa accctggcac tgcgccgaaa tatggcatcc
gtggtatccc gactctgctg 240ctgttcaaaa acggtgaaca ccaccaccac caccactaa
27910279DNAArtificialModified truncated E. coli
thioredoxin ORF lacking lysine with C-terminal his tag 10atgagcgata
aaattattca cctgactgac gacagttttg acacggatgt actcaaagcg 60gacggggcga
tcctcgtcga tttctgggca gagtggtgcg gtccgtgcaa aatgatcgcc 120ccgattctgg
atgaaatcgc tgacgaatat cagggcaaac tgaccgttgc aaaactgaac 180atcgatcaaa
accctggcac tgcgccgaaa tatggcatcc gtggtatccc gactctgctg 240ctgttcaaaa
acggtgaaca ccaccaccac caccactaa
2791192PRTArtificialTruncated E. coli thioredoxin ORF with
C-terminal his tag 11Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe
Asp Thr Asp1 5 10 15Val
Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp20
25 30Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu
Asp Glu Ile Ala Asp35 40 45Glu Tyr Gln
Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn50 55
60Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro
Thr Leu Leu65 70 75
80Leu Phe Lys Asn Gly Glu His His His His His His85
901292PRTArtificialModified truncated E. coli thioredoxin ORF
lacking lysine with C-terminal his tag 12Met Ser Asp Arg Ile Ile His Leu
Thr Asp Asp Ser Phe Asp Thr Asp1 5 10
15Val Leu Arg Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala
Glu Trp20 25 30Cys Gly Pro Arg Met Cys
Ile Ala Pro Ile Leu Asp Glu Arg Ala Asp35 40
45Glu Tyr Gln Gly Arg Leu Thr Val Ala Arg Leu Asn Ile Asp Gln Asn50
55 60Pro Gly Thr Ala Pro Arg Tyr Gly Ile
Arg Gly Ile Pro Thr Leu Leu65 70 75
80Leu Phe Arg Asn Gly Glu His His His His His His85
90131595DNAArtificialBH 6mer ORF having six repeats of modified
truncated E. coli thioredoxin ORF lacking lysine and C-terminal
his tag 13agatctatgt gtgatcgtat tattcacctg actgacgact gctttgacac
ggatgtactc 60cgcgcggacg gggcgcgtct cgtcgatttc tgggcagagt ggtgcggtcc
gcgtatgtgt 120atcgccccga ttctggatga acgtgctgac gaatatcagg gccgcctgac
cgttgcacgt 180ctgaacatcg atcaaaaccc tggcactagc gctatgtgtg atcgtattat
tcatctgact 240gatgactgct ttgacacgga cgtactccgt gcggacgggg cgcgtctcgt
cgatttctgg 300gcagagtggt gcggtccacg tatgtgtatc gcgccgattc tggatgaacg
tgcagacgaa 360tatcaaggcc gcctgaccgt tgcacgcctg aacatcgatc aaaaccctgg
tactgcgccg 420cgctatggca tccgtggcat cccgactctg ctgctgttcc gtaacggcga
aggtaccatg 480tgtgaccgta ttatccacct gactgacgac tgcttcgaca cggatgtact
ccgcgcggat 540ggggcgcgtc tcgtcgactt ctgggcagag tggtgcggtc ctcgtatgtg
tatcgcccct 600attctggatg agcgtgctga cgaatatcag ggtcgcctga ccgttgcacg
tctgaatatc 660gatcaaaacc ctggcactgc accgcgctat ggcatccgtg gtatcccgac
tctcctgctg 720ttccgtaacg gcgaagaatt catgtgtgat cgtatcattc acctgactga
cgactgtttt 780gacacggatg ttctccgcgc ggacggggcg cgtctcgtag atttctgggc
agagtggtgc 840ggcccgcgta tgtgtatcgc cccgattctc gatgaacgtg ctgacgaata
tcagggtcgc 900ctgaccgttg cccgtctgaa catcgatcaa aaccctggca ctgcaccgcg
ctatggcatc 960cgtggtatcc caactctgct gctgttccgt aacggcgaaa ccggtatgtg
cgatcgcatt 1020attcacctga ctgatgactg ctttgacacg gacgtactcc gcgcggacgg
ggcgcgcctc 1080gtcgatttct gggcagagtg gtgcggtccg cgtatgtgta tcgcgccgat
tctggatgaa 1140cgtgcggacg aatatcaggg ccgcctgact gttgcacgtc tgaacatcga
ccaaaaccct 1200ggcactgcgc ctcgctatgg catccgtggt atcccgactc tgctgctctt
ccgtaacggc 1260gaagccggca tgtgtgatcg tatcattcac ctgactgatg actgcttcga
cacggatgta 1320ctccgcgccg acggggcgat cctcgtcgat ttctgggcag aatggtgcgg
tccgcgtatg 1380tgtatcgctc cgatcctgga tgaaatcgct gatgaatatc agggccgcct
caccgttgca 1440cgtctgaata tcgatcaaaa ccctggtact gcgccgcgct atggtatccg
tggcatcccg 1500actcttctgc ttttccgtaa cggcgaagcc ggcaccggtg aattcggtac
cagcgctcac 1560caccaccacc accaccatca tcatcacgtt taaac
159514532PRTArtificialpTRC BH 60 kDa expression product 14Met
His Gly Ser Met Cys Asp Arg Ile Ile His Leu Thr Asp Asp Cys1
5 10 15Phe Asp Thr Asp Val Leu Arg
Ala Asp Gly Ala Arg Leu Val Asp Phe20 25
30Trp Ala Glu Trp Cys Gly Pro Arg Met Cys Ile Ala Pro Ile Leu Asp35
40 45Glu Arg Ala Asp Glu Tyr Gln Gly Arg Leu
Thr Val Ala Arg Leu Asn50 55 60Ile Asp
Gln Asn Pro Gly Thr Ser Ala Met Cys Asp Arg Ile Ile His65
70 75 80Leu Thr Asp Asp Cys Phe Asp
Thr Asp Val Leu Arg Ala Asp Gly Ala85 90
95Arg Leu Val Asp Phe Trp Ala Glu Trp Cys Gly Pro Arg Met Cys Ile100
105 110Ala Pro Ile Leu Asp Glu Arg Ala Asp
Glu Tyr Gln Gly Arg Leu Thr115 120 125Val
Ala Arg Leu Asn Ile Asp Gln Asn Pro Gly Thr Ala Pro Arg Tyr130
135 140Gly Ile Arg Gly Ile Pro Thr Leu Leu Leu Phe
Arg Asn Gly Glu Gly145 150 155
160Thr Met Cys Asp Arg Ile Ile His Leu Thr Asp Asp Cys Phe Asp
Thr165 170 175Asp Val Leu Arg Ala Asp Gly
Ala Arg Leu Val Asp Phe Trp Ala Glu180 185
190Trp Cys Gly Pro Arg Met Cys Ile Ala Pro Ile Leu Asp Glu Arg Ala195
200 205Asp Glu Tyr Gln Gly Arg Leu Thr Val
Ala Arg Leu Asn Ile Asp Gln210 215 220Asn
Pro Gly Thr Ala Pro Arg Tyr Gly Ile Arg Gly Ile Pro Thr Leu225
230 235 240Leu Leu Phe Arg Asn Gly
Glu Glu Phe Met Cys Asp Arg Ile Ile His245 250
255Leu Thr Asp Asp Cys Phe Asp Thr Asp Val Leu Arg Ala Asp Gly
Ala260 265 270Arg Leu Val Asp Phe Trp Ala
Glu Trp Cys Gly Pro Arg Met Cys Ile275 280
285Ala Pro Ile Leu Asp Glu Arg Ala Asp Glu Tyr Gln Gly Arg Leu Thr290
295 300Val Ala Arg Leu Asn Ile Asp Gln Asn
Pro Gly Thr Ala Pro Arg Tyr305 310 315
320Gly Ile Arg Gly Ile Pro Thr Leu Leu Leu Phe Arg Asn Gly
Glu Thr325 330 335Gly Met Cys Asp Arg Ile
Ile His Leu Thr Asp Asp Cys Phe Asp Thr340 345
350Asp Val Leu Arg Ala Asp Gly Ala Arg Leu Val Asp Phe Trp Ala
Glu355 360 365Trp Cys Gly Pro Arg Met Cys
Ile Ala Pro Ile Leu Asp Glu Arg Ala370 375
380Asp Glu Tyr Gln Gly Arg Leu Thr Val Ala Arg Leu Asn Ile Asp Gln385
390 395 400Asn Pro Gly Thr
Ala Pro Arg Tyr Gly Ile Arg Gly Ile Pro Thr Leu405 410
415Leu Leu Phe Arg Asn Gly Glu Ala Gly Met Cys Asp Arg Ile
Ile His420 425 430Leu Thr Asp Asp Cys Phe
Asp Thr Asp Val Leu Arg Ala Asp Gly Ala435 440
445Ile Leu Val Asp Phe Trp Ala Glu Trp Cys Gly Pro Arg Met Cys
Ile450 455 460Ala Pro Ile Leu Asp Glu Ile
Ala Asp Glu Tyr Gln Gly Arg Leu Thr465 470
475 480Val Ala Arg Leu Asn Ile Asp Gln Asn Pro Gly Thr
Ala Pro Arg Tyr485 490 495Gly Ile Arg Gly
Ile Pro Thr Leu Leu Leu Phe Arg Asn Gly Glu Ala500 505
510Gly Thr Gly Glu Phe Gly Thr Ser Ala His His His His His
His His515 520 525His His His
Val53015264PRTArtificialpTRC BH 30 kDa expression product 15Met His Gly
Ser Met Cys Asp Arg Ile Ile His Leu Thr Asp Asp Cys1 5
10 15Phe Asp Thr Asp Val Leu Arg Ala Asp
Gly Ala Arg Leu Val Asp Phe20 25 30Trp
Ala Glu Trp Cys Gly Pro Arg Met Cys Ile Ala Pro Ile Leu Asp35
40 45Glu Arg Ala Asp Glu Tyr Gln Gly Arg Leu Thr
Val Ala Arg Leu Asn50 55 60Ile Asp Gln
Asn Pro Gly Thr Ser Ala Met Cys Asp Arg Ile Ile His65 70
75 80Leu Thr Asp Asp Cys Phe Asp Thr
Asp Val Leu Arg Ala Asp Gly Ala85 90
95Arg Leu Val Asp Phe Trp Ala Glu Trp Cys Gly Pro Arg Met Cys Ile100
105 110Ala Pro Ile Leu Asp Glu Arg Ala Asp Glu
Tyr Gln Gly Arg Leu Thr115 120 125Val Ala
Arg Leu Asn Ile Asp Gln Asn Pro Gly Thr Ala Pro Arg Tyr130
135 140Gly Ile Arg Gly Ile Pro Thr Leu Leu Leu Phe Arg
Asn Gly Glu Gly145 150 155
160Thr Met Cys Asp Arg Ile Ile His Leu Thr Asp Asp Cys Phe Asp Thr165
170 175Asp Val Leu Arg Ala Asp Gly Ala Arg
Leu Val Asp Phe Trp Ala Glu180 185 190Trp
Cys Gly Pro Arg Met Cys Ile Ala Pro Ile Leu Asp Glu Arg Ala195
200 205Asp Glu Tyr Gln Gly Arg Leu Thr Val Ala Arg
Leu Asn Ile Asp Gln210 215 220Asn Pro Gly
Thr Ala Pro Arg Tyr Gly Ile Arg Gly Ile Pro Thr Leu225
230 235 240Leu Leu Phe Arg Asn Gly Glu
Glu Phe Gly Thr Ser Ala His His His245 250
255His His His His His His His Val26016354PRTArtificialpTRC BH 40 kDa
expression product 16Met His Gly Ser Met Cys Asp Arg Ile Ile His Leu Thr
Asp Asp Cys1 5 10 15Phe
Asp Thr Asp Val Leu Arg Ala Asp Gly Ala Arg Leu Val Asp Phe20
25 30Trp Ala Glu Trp Cys Gly Pro Arg Met Cys Ile
Ala Pro Ile Leu Asp35 40 45Glu Arg Ala
Asp Glu Tyr Gln Gly Arg Leu Thr Val Ala Arg Leu Asn50 55
60Ile Asp Gln Asn Pro Gly Thr Ser Ala Met Cys Asp Arg
Ile Ile His65 70 75
80Leu Thr Asp Asp Cys Phe Asp Thr Asp Val Leu Arg Ala Asp Gly Ala85
90 95Arg Leu Val Asp Phe Trp Ala Glu Trp Cys
Gly Pro Arg Met Cys Ile100 105 110Ala Pro
Ile Leu Asp Glu Arg Ala Asp Glu Tyr Gln Gly Arg Leu Thr115
120 125Val Ala Arg Leu Asn Ile Asp Gln Asn Pro Gly Thr
Ala Pro Arg Tyr130 135 140Gly Ile Arg Gly
Ile Pro Thr Leu Leu Leu Phe Arg Asn Gly Glu Gly145 150
155 160Thr Met Cys Asp Arg Ile Ile His Leu
Thr Asp Asp Cys Phe Asp Thr165 170 175Asp
Val Leu Arg Ala Asp Gly Ala Arg Leu Val Asp Phe Trp Ala Glu180
185 190Trp Cys Gly Pro Arg Met Cys Ile Ala Pro Ile
Leu Asp Glu Arg Ala195 200 205Asp Glu Tyr
Gln Gly Arg Leu Thr Val Ala Arg Leu Asn Ile Asp Gln210
215 220Asn Pro Gly Thr Ala Pro Arg Tyr Gly Ile Arg Gly
Ile Pro Thr Leu225 230 235
240Leu Leu Phe Arg Asn Gly Glu Glu Phe Met Cys Asp Arg Ile Ile His245
250 255Leu Thr Asp Asp Cys Phe Asp Thr Asp
Val Leu Arg Ala Asp Gly Ala260 265 270Arg
Leu Val Asp Phe Trp Ala Glu Trp Cys Gly Pro Arg Met Cys Ile275
280 285Ala Pro Ile Leu Asp Glu Arg Ala Asp Glu Tyr
Gln Gly Arg Leu Thr290 295 300Val Ala Arg
Leu Asn Ile Asp Gln Asn Pro Gly Thr Ala Pro Arg Tyr305
310 315 320Gly Ile Arg Gly Ile Pro Thr
Leu Leu Leu Phe Arg Asn Gly Glu Thr325 330
335Gly Glu Phe Gly Thr Ser Ala His His His His His His His His His340
345 350His Val17444PRTArtificialpTRC BH 50
kDa expression product 17Met His Gly Ser Met Cys Asp Arg Ile Ile His Leu
Thr Asp Asp Cys1 5 10
15Phe Asp Thr Asp Val Leu Arg Ala Asp Gly Ala Arg Leu Val Asp Phe20
25 30Trp Ala Glu Trp Cys Gly Pro Arg Met Cys
Ile Ala Pro Ile Leu Asp35 40 45Glu Arg
Ala Asp Glu Tyr Gln Gly Arg Leu Thr Val Ala Arg Leu Asn50
55 60Ile Asp Gln Asn Pro Gly Thr Ser Ala Met Cys Asp
Arg Ile Ile His65 70 75
80Leu Thr Asp Asp Cys Phe Asp Thr Asp Val Leu Arg Ala Asp Gly Ala85
90 95Arg Leu Val Asp Phe Trp Ala Glu Trp Cys
Gly Pro Arg Met Cys Ile100 105 110Ala Pro
Ile Leu Asp Glu Arg Ala Asp Glu Tyr Gln Gly Arg Leu Thr115
120 125Val Ala Arg Leu Asn Ile Asp Gln Asn Pro Gly Thr
Ala Pro Arg Tyr130 135 140Gly Ile Arg Gly
Ile Pro Thr Leu Leu Leu Phe Arg Asn Gly Glu Gly145 150
155 160Thr Met Cys Asp Arg Ile Ile His Leu
Thr Asp Asp Cys Phe Asp Thr165 170 175Asp
Val Leu Arg Ala Asp Gly Ala Arg Leu Val Asp Phe Trp Ala Glu180
185 190Trp Cys Gly Pro Arg Met Cys Ile Ala Pro Ile
Leu Asp Glu Arg Ala195 200 205Asp Glu Tyr
Gln Gly Arg Leu Thr Val Ala Arg Leu Asn Ile Asp Gln210
215 220Asn Pro Gly Thr Ala Pro Arg Tyr Gly Ile Arg Gly
Ile Pro Thr Leu225 230 235
240Leu Leu Phe Arg Asn Gly Glu Glu Phe Met Cys Asp Arg Ile Ile His245
250 255Leu Thr Asp Asp Cys Phe Asp Thr Asp
Val Leu Arg Ala Asp Gly Ala260 265 270Arg
Leu Val Asp Phe Trp Ala Glu Trp Cys Gly Pro Arg Met Cys Ile275
280 285Ala Pro Ile Leu Asp Glu Arg Ala Asp Glu Tyr
Gln Gly Arg Leu Thr290 295 300Val Ala Arg
Leu Asn Ile Asp Gln Asn Pro Gly Thr Ala Pro Arg Tyr305
310 315 320Gly Ile Arg Gly Ile Pro Thr
Leu Leu Leu Phe Arg Asn Gly Glu Thr325 330
335Gly Met Cys Asp Arg Ile Ile His Leu Thr Asp Asp Cys Phe Asp Thr340
345 350Asp Val Leu Arg Ala Asp Gly Ala Arg
Leu Val Asp Phe Trp Ala Glu355 360 365Trp
Cys Gly Pro Arg Met Cys Ile Ala Pro Ile Leu Asp Glu Arg Ala370
375 380Asp Glu Tyr Gln Gly Arg Leu Thr Val Ala Arg
Leu Asn Ile Asp Gln385 390 395
400Asn Pro Gly Thr Ala Pro Arg Tyr Gly Ile Arg Gly Ile Pro Thr
Leu405 410 415Leu Leu Phe Arg Asn Gly Glu
Ala Gly Thr Gly Glu Phe Gly Thr Ser420 425
430Ala His His His His His His His His His His Val435
4401821DNAArtificialpTrCHisFOR forward primer 18gaggtatata ttaatgtatc g
211918DNAArtificialpBAD_Rev
reverse primer 19gatttaatct gtatcagg
182030DNAArtificial50.1_F forward primer 20ccggagatct
atgtgtgatc gtattattca
302130DNAArtificial50.1_R reverse primer 21ccggctcgag ttcgccgtta
cggaaaagca 302236DNAArtificial50.2_F
forward primer 22ccggctcgag atgtgtgatc gtattattca tctgac
362330DNAArtificial50.2_R reverse primer 23ccggcctagg
ttcgccgtta cggaaaagca
302462DNAArtificial50.2_10HIS-Pme_R reverse primer 24gtttaaacgt
gatgatgatg gtggtggtgg tggtggtgtt cgccgttacg gaaaagcaga 60ag
622536DNAArtificial50.3_F forward primer 25ccggcctagg atgtgtgatc
gtattattca tctgac 362630DNAArtificial50.3_R
reverse primer 26ccggcggccg ttcgccgtta cggaaaagca
302762DNAArtificial50.3_10HIS-Pme_R reverse primer
27gtttaaacgt gatgatgatg gtggtggtgg tggtggtgtt cgccgttacg gaaaagcaga
60ag
622831DNAArtificial50.4_F forward primer 28ccggcggccg atgtgtgatc
gtattattca t
312962DNAArtificial50.4_10HIS-Pme_R reverse primer 29gtttaaacgt
gatgatgatg gtggtggtgg tggtggtgtt cgccgttacg gaaaagcaga 60ag
623018DNAArtificialTA50kd_1F primer 30gtgcggtcca cgtatgtg
183116DNAArtificialTA50kd_2F primer
31ggcgcgtctc gtcgac
163219DNAArtificialTA50kd_2R primer 32actctgccca gaagtcgac
193317DNAArtificialTA50kd_3F primer
33cgaaaccggt atgtgcg
173416DNAArtificialTA50kd_3R primer 34cgatcgcaca taccgg
163520DNAArtificialT7#6 primer
35taatacgact cactataggg
203620DNAArtificialPCRII200F#738 primer 36cacacaggaa acagctatga
20371314DNAArtificialpTRC BH 50 kDa
ORF 37atgtgtgatc gtattattca tctgactgat gactgctttg acacggacgt actccgtgcg
60gacggggcgc gtctcgtcga tttctgggca gagtggtgcg gtccacgtat gtgtatcgcg
120ccgattctgg atgaacgtgc agacgaatat caaggccgcc tgaccgttgc acgcctgaac
180atcgatcaaa accctggtac tgcgccgcgc tatggcatcc gtggcatccc gactctgctg
240ctgttccgta acggcgaagg taccatgtgt gaccgtatta tccacctgac tgacgactgc
300ttcgacacgg atgtactccg cgcggatggg gcgcgtctcg tcgacttctg ggcagagtgg
360tgcggtcctc gtatgtgtat cgcccctatt ctggatgagc gtgctgacga atatcagggt
420cgcctgaccg ttgcacgtct gaatatcgat caaaaccctg gcactgcacc gcgctatggc
480atccgtggta tcccgactct cctgctgttc cgtaacggcg aagaattcat gtgtgatcgt
540atcattcacc tgactgacga ctgttttgac acggatgttc tccgcgcgga cggggcgcgt
600ctcgtagatt tctgggcaga gtggtgcggc ccgcgtatgt gtatcgcccc gattctcgat
660gaacgtgctg acgaatatca gggtcgcctg accgttgccc gtctgaacat cgatcaaaac
720cctggcactg caccgcgcta tggcatccgt ggtatcccaa ctctgctgct gttccgtaac
780ggcgaaaccg gtatgtgcga tcgcattatt cacctgactg atgactgctt tgacacggac
840gtactccgcg cggacggggc gcgcctcgtc gatttctggg cagagtggtg cggtccgcgt
900atgtgtatcg cgccgattct ggatgaacgt gcggacgaat atcagggccg cctgactgtt
960gcacgtctga acatcgacca aaaccctggc actgcgcctc gctatggcat ccgtggtatc
1020ccgactctgc tgctcttccg taacggcgaa gccggcatgt gtgatcgtat cattcacctg
1080actgatgact gcttcgacac ggatgtactc cgcgccgacg gggcgatcct cgtcgatttc
1140tgggcagaat ggtgcggtcc gcgtatgtgt atcgctccga tcctggatga aatcgctgat
1200gaatatcagg gccgcctcac cgttgcacgt ctgaatatcg atcaaaaccc tggtactgcg
1260ccgcgctatg gtatccgtgg catcccgact cttctgcttt tccgtaacgg cgaa
131438893PRTArtificialpTRC 110 kDa expression product 38Met His Gly Ser
Met Cys Asp Arg Ile Ile His Leu Thr Asp Asp Cys1 5
10 15Phe Asp Thr Asp Val Leu Arg Ala Asp Gly
Ala Arg Leu Val Asp Phe20 25 30Trp Ala
Glu Trp Cys Gly Pro Arg Met Cys Ile Ala Pro Ile Leu Asp35
40 45Glu Arg Ala Asp Glu Tyr Gln Gly Arg Leu Thr Val
Ala Arg Leu Asn50 55 60Ile Asp Gln Asn
Pro Gly Thr Ala Pro Arg Tyr Gly Ile Arg Gly Ile65 70
75 80Pro Thr Leu Leu Leu Phe Arg Asn Gly
Glu Gly Thr Met Cys Asp Arg85 90 95Ile
Ile His Leu Thr Asp Asp Cys Phe Asp Thr Asp Val Leu Arg Ala100
105 110Asp Gly Ala Arg Leu Val Asp Phe Trp Ala Glu
Trp Cys Gly Pro Arg115 120 125Met Cys Ile
Ala Pro Ile Leu Asp Glu Arg Ala Asp Glu Tyr Gln Gly130
135 140Arg Leu Thr Val Ala Arg Leu Asn Ile Asp Gln Asn
Pro Gly Thr Ala145 150 155
160Pro Arg Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu Leu Phe Arg Asn165
170 175Gly Glu Glu Phe Met Cys Asp Arg Ile
Ile His Leu Thr Asp Asp Cys180 185 190Phe
Asp Thr Asp Val Leu Arg Ala Asp Gly Ala Arg Leu Val Asp Phe195
200 205Trp Ala Glu Trp Cys Gly Pro Arg Met Cys Ile
Ala Pro Ile Leu Asp210 215 220Glu Arg Ala
Asp Glu Tyr Gln Gly Arg Leu Thr Val Ala Arg Leu Asn225
230 235 240Ile Asp Gln Asn Pro Gly Thr
Ala Pro Arg Tyr Gly Ile Arg Gly Ile245 250
255Pro Thr Leu Leu Leu Phe Arg Asn Gly Glu Thr Gly Met Cys Asp Arg260
265 270Ile Ile His Leu Thr Asp Asp Cys Phe
Asp Thr Asp Val Leu Arg Ala275 280 285Asp
Gly Ala Arg Leu Val Asp Phe Trp Ala Glu Trp Cys Gly Pro Arg290
295 300Met Cys Ile Ala Pro Ile Leu Asp Glu Arg Ala
Asp Glu Tyr Gln Gly305 310 315
320Arg Leu Thr Val Ala Arg Leu Asn Ile Asp Gln Asn Pro Gly Thr
Ala325 330 335Pro Arg Tyr Gly Ile Arg Gly
Ile Pro Thr Leu Leu Leu Phe Arg Asn340 345
350Gly Glu Ala Gly Met Cys Asp Arg Ile Ile His Leu Thr Asp Asp Cys355
360 365Phe Asp Thr Asp Val Leu Arg Ala Asp
Gly Ala Ile Leu Val Asp Phe370 375 380Trp
Ala Glu Trp Cys Gly Pro Arg Met Cys Ile Ala Pro Ile Leu Asp385
390 395 400Glu Ile Ala Asp Glu Tyr
Gln Gly Arg Leu Thr Val Ala Arg Leu Asn405 410
415Ile Asp Gln Asn Pro Gly Thr Ala Pro Arg Tyr Gly Ile Arg Gly
Ile420 425 430Pro Thr Leu Leu Leu Phe Arg
Asn Gly Glu Leu Glu Met Cys Asp Arg435 440
445Ile Ile His Leu Thr Asp Asp Cys Phe Asp Thr Asp Val Leu Arg Ala450
455 460Asp Gly Ala Arg Leu Val Asp Phe Trp
Ala Glu Trp Cys Gly Pro Arg465 470 475
480Met Cys Ile Ala Pro Ile Leu Asp Glu Arg Ala Asp Glu Tyr
Gln Gly485 490 495Arg Leu Thr Val Ala Arg
Leu Asn Ile Asp Gln Asn Pro Gly Thr Ala500 505
510Pro Arg Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu Leu Phe Arg
Asn515 520 525Gly Glu Gly Thr Met Cys Asp
Arg Ile Ile His Leu Thr Asp Asp Cys530 535
540Phe Asp Thr Asp Val Leu Arg Ala Asp Gly Ala Arg Leu Val Asp Phe545
550 555 560Trp Ala Glu Trp
Cys Gly Pro Arg Met Cys Ile Ala Pro Ile Leu Asp565 570
575Glu Arg Ala Asp Glu Tyr Gln Gly Arg Leu Thr Val Ala Arg
Leu Asn580 585 590Ile Asp Gln Asn Pro Gly
Thr Ala Pro Arg Tyr Gly Ile Arg Gly Ile595 600
605Pro Thr Leu Leu Leu Phe Arg Asn Gly Glu Glu Phe Met Cys Asp
Arg610 615 620Ile Ile His Leu Thr Asp Asp
Cys Phe Asp Thr Asp Val Leu Arg Ala625 630
635 640Asp Gly Ala Arg Leu Val Asp Phe Trp Ala Glu Trp
Cys Gly Pro Arg645 650 655Met Cys Ile Ala
Pro Ile Leu Asp Glu Arg Ala Asp Glu Tyr Gln Gly660 665
670Arg Leu Thr Val Ala Arg Leu Asn Ile Asp Gln Asn Pro Gly
Thr Ala675 680 685Pro Arg Tyr Gly Ile Arg
Gly Ile Pro Thr Leu Leu Leu Phe Arg Asn690 695
700Gly Glu Thr Gly Met Cys Asp Arg Ile Ile His Leu Thr Asp Asp
Cys705 710 715 720Phe Asp
Thr Asp Val Leu Arg Ala Asp Gly Ala Arg Leu Val Asp Phe725
730 735Trp Ala Glu Trp Cys Gly Pro Arg Met Cys Ile Ala
Pro Ile Leu Asp740 745 750Glu Arg Ala Asp
Glu Tyr Gln Gly Arg Leu Thr Val Ala Arg Leu Asn755 760
765Ile Asp Gln Asn Pro Gly Thr Ala Pro Arg Tyr Gly Ile Arg
Gly Ile770 775 780Pro Thr Leu Leu Leu Phe
Arg Asn Gly Glu Ala Gly Met Cys Asp Arg785 790
795 800Ile Ile His Leu Thr Asp Asp Cys Phe Asp Thr
Asp Val Leu Arg Ala805 810 815Asp Gly Ala
Ile Leu Val Asp Phe Trp Ala Glu Trp Cys Gly Pro Arg820
825 830Met Cys Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp
Glu Tyr Gln Gly835 840 845Arg Leu Thr Val
Ala Arg Leu Asn Ile Asp Gln Asn Pro Gly Thr Ala850 855
860Pro Arg Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu Leu Phe
Arg Asn865 870 875 880Gly
Glu His His His His His His His His His His Val885
890391333PRTArtificialpTRC 160 kDa predicted expression product 39Met His
Gly Ser Met Cys Asp Arg Ile Ile His Leu Thr Asp Asp Cys1 5
10 15Phe Asp Thr Asp Val Leu Arg Ala
Asp Gly Ala Arg Leu Val Asp Phe20 25
30Trp Ala Glu Trp Cys Gly Pro Arg Met Cys Ile Ala Pro Ile Leu Asp35
40 45Glu Arg Ala Asp Glu Tyr Gln Gly Arg Leu
Thr Val Ala Arg Leu Asn50 55 60Ile Asp
Gln Asn Pro Gly Thr Ala Pro Arg Tyr Gly Ile Arg Gly Ile65
70 75 80Pro Thr Leu Leu Leu Phe Arg
Asn Gly Glu Gly Thr Met Cys Asp Arg85 90
95Ile Ile His Leu Thr Asp Asp Cys Phe Asp Thr Asp Val Leu Arg Ala100
105 110Asp Gly Ala Arg Leu Val Asp Phe Trp
Ala Glu Trp Cys Gly Pro Arg115 120 125Met
Cys Ile Ala Pro Ile Leu Asp Glu Arg Ala Asp Glu Tyr Gln Gly130
135 140Arg Leu Thr Val Ala Arg Leu Asn Ile Asp Gln
Asn Pro Gly Thr Ala145 150 155
160Pro Arg Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu Leu Phe Arg
Asn165 170 175Gly Glu Glu Phe Met Cys Asp
Arg Ile Ile His Leu Thr Asp Asp Cys180 185
190Phe Asp Thr Asp Val Leu Arg Ala Asp Gly Ala Arg Leu Val Asp Phe195
200 205Trp Ala Glu Trp Cys Gly Pro Arg Met
Cys Ile Ala Pro Ile Leu Asp210 215 220Glu
Arg Ala Asp Glu Tyr Gln Gly Arg Leu Thr Val Ala Arg Leu Asn225
230 235 240Ile Asp Gln Asn Pro Gly
Thr Ala Pro Arg Tyr Gly Ile Arg Gly Ile245 250
255Pro Thr Leu Leu Leu Phe Arg Asn Gly Glu Thr Gly Met Cys Asp
Arg260 265 270Ile Ile His Leu Thr Asp Asp
Cys Phe Asp Thr Asp Val Leu Arg Ala275 280
285Asp Gly Ala Arg Leu Val Asp Phe Trp Ala Glu Trp Cys Gly Pro Arg290
295 300Met Cys Ile Ala Pro Ile Leu Asp Glu
Arg Ala Asp Glu Tyr Gln Gly305 310 315
320Arg Leu Thr Val Ala Arg Leu Asn Ile Asp Gln Asn Pro Gly
Thr Ala325 330 335Pro Arg Tyr Gly Ile Arg
Gly Ile Pro Thr Leu Leu Leu Phe Arg Asn340 345
350Gly Glu Ala Gly Met Cys Asp Arg Ile Ile His Leu Thr Asp Asp
Cys355 360 365Phe Asp Thr Asp Val Leu Arg
Ala Asp Gly Ala Ile Leu Val Asp Phe370 375
380Trp Ala Glu Trp Cys Gly Pro Arg Met Cys Ile Ala Pro Ile Leu Asp385
390 395 400Glu Ile Ala Asp
Glu Tyr Gln Gly Arg Leu Thr Val Ala Arg Leu Asn405 410
415Ile Asp Gln Asn Pro Gly Thr Ala Pro Arg Tyr Gly Ile Arg
Gly Ile420 425 430Pro Thr Leu Leu Leu Phe
Arg Asn Gly Glu Leu Glu Met Cys Asp Arg435 440
445Ile Ile His Leu Thr Asp Asp Cys Phe Asp Thr Asp Val Leu Arg
Ala450 455 460Asp Gly Ala Arg Leu Val Asp
Phe Trp Ala Glu Trp Cys Gly Pro Arg465 470
475 480Met Cys Ile Ala Pro Ile Leu Asp Glu Arg Ala Asp
Glu Tyr Gln Gly485 490 495Arg Leu Thr Val
Ala Arg Leu Asn Ile Asp Gln Asn Pro Gly Thr Ala500 505
510Pro Arg Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu Leu Phe
Arg Asn515 520 525Gly Glu Gly Thr Met Cys
Asp Arg Ile Ile His Leu Thr Asp Asp Cys530 535
540Phe Asp Thr Asp Val Leu Arg Ala Asp Gly Ala Arg Leu Val Asp
Phe545 550 555 560Trp Ala
Glu Trp Cys Gly Pro Arg Met Cys Ile Ala Pro Ile Leu Asp565
570 575Glu Arg Ala Asp Glu Tyr Gln Gly Arg Leu Thr Val
Ala Arg Leu Asn580 585 590Ile Asp Gln Asn
Pro Gly Thr Ala Pro Arg Tyr Gly Ile Arg Gly Ile595 600
605Pro Thr Leu Leu Leu Phe Arg Asn Gly Glu Glu Phe Met Cys
Asp Arg610 615 620Ile Ile His Leu Thr Asp
Asp Cys Phe Asp Thr Asp Val Leu Arg Ala625 630
635 640Asp Gly Ala Arg Leu Val Asp Phe Trp Ala Glu
Trp Cys Gly Pro Arg645 650 655Met Cys Ile
Ala Pro Ile Leu Asp Glu Arg Ala Asp Glu Tyr Gln Gly660
665 670Arg Leu Thr Val Ala Arg Leu Asn Ile Asp Gln Asn
Pro Gly Thr Ala675 680 685Pro Arg Tyr Gly
Ile Arg Gly Ile Pro Thr Leu Leu Leu Phe Arg Asn690 695
700Gly Glu Thr Gly Met Cys Asp Arg Ile Ile His Leu Thr Asp
Asp Cys705 710 715 720Phe
Asp Thr Asp Val Leu Arg Ala Asp Gly Ala Arg Leu Val Asp Phe725
730 735Trp Ala Glu Trp Cys Gly Pro Arg Met Cys Ile
Ala Pro Ile Leu Asp740 745 750Glu Arg Ala
Asp Glu Tyr Gln Gly Arg Leu Thr Val Ala Arg Leu Asn755
760 765Ile Asp Gln Asn Pro Gly Thr Ala Pro Arg Tyr Gly
Ile Arg Gly Ile770 775 780Pro Thr Leu Leu
Leu Phe Arg Asn Gly Glu Ala Gly Met Cys Asp Arg785 790
795 800Ile Ile His Leu Thr Asp Asp Cys Phe
Asp Thr Asp Val Leu Arg Ala805 810 815Asp
Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp Cys Gly Pro Arg820
825 830Met Cys Ile Ala Pro Ile Leu Asp Glu Ile Ala
Asp Glu Tyr Gln Gly835 840 845Arg Leu Thr
Val Ala Arg Leu Asn Ile Asp Gln Asn Pro Gly Thr Ala850
855 860Pro Arg Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu
Leu Phe Arg Asn865 870 875
880Gly Glu Pro Arg Met Cys Asp Arg Ile Ile His Leu Thr Asp Asp Cys885
890 895Phe Asp Thr Asp Val Leu Arg Ala Asp
Gly Ala Arg Leu Val Asp Phe900 905 910Trp
Ala Glu Trp Cys Gly Pro Arg Met Cys Ile Ala Pro Ile Leu Asp915
920 925Glu Arg Ala Asp Glu Tyr Gln Gly Arg Leu Thr
Val Ala Arg Leu Asn930 935 940Ile Asp Gln
Asn Pro Gly Thr Ala Pro Arg Tyr Gly Ile Arg Gly Ile945
950 955 960Pro Thr Leu Leu Leu Phe Arg
Asn Gly Glu Gly Thr Met Cys Asp Arg965 970
975Ile Ile His Leu Thr Asp Asp Cys Phe Asp Thr Asp Val Leu Arg Ala980
985 990Asp Gly Ala Arg Leu Val Asp Phe Trp
Ala Glu Trp Cys Gly Pro Arg995 1000
1005Met Cys Ile Ala Pro Ile Leu Asp Glu Arg Ala Asp Glu Tyr Gln1010
1015 1020Gly Arg Leu Thr Val Ala Arg Leu
Asn Ile Asp Gln Asn Pro Gly1025 1030
1035Thr Ala Pro Arg Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu Leu1040
1045 1050Phe Arg Asn Gly Glu Glu Phe Met
Cys Asp Arg Ile Ile His Leu1055 1060
1065Thr Asp Asp Cys Phe Asp Thr Asp Val Leu Arg Ala Asp Gly Ala1070
1075 1080Arg Leu Val Asp Phe Trp Ala Glu
Trp Cys Gly Pro Arg Met Cys1085 1090
1095Ile Ala Pro Ile Leu Asp Glu Arg Ala Asp Glu Tyr Gln Gly Arg1100
1105 1110Leu Thr Val Ala Arg Leu Asn Ile
Asp Gln Asn Pro Gly Thr Ala1115 1120
1125Pro Arg Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu Leu Phe Arg1130
1135 1140Asn Gly Glu Thr Gly Met Cys Asp
Arg Ile Ile His Leu Thr Asp1145 1150
1155Asp Cys Phe Asp Thr Asp Val Leu Arg Ala Asp Gly Ala Arg Leu1160
1165 1170Val Asp Phe Trp Ala Glu Trp Cys
Gly Pro Arg Met Cys Ile Ala1175 1180
1185Pro Ile Leu Asp Glu Arg Ala Asp Glu Tyr Gln Gly Arg Leu Thr1190
1195 1200Val Ala Arg Leu Asn Ile Asp Gln
Asn Pro Gly Thr Ala Pro Arg1205 1210
1215Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu Leu Phe Arg Asn Gly1220
1225 1230Glu Ala Gly Met Cys Asp Arg Ile
Ile His Leu Thr Asp Asp Cys1235 1240
1245Phe Asp Thr Asp Val Leu Arg Ala Asp Gly Ala Ile Leu Val Asp1250
1255 1260Phe Trp Ala Glu Trp Cys Gly Pro
Arg Met Cys Ile Ala Pro Ile1265 1270
1275Leu Asp Glu Ile Ala Asp Glu Tyr Gln Gly Arg Leu Thr Val Ala1280
1285 1290Arg Leu Asn Ile Asp Gln Asn Pro
Gly Thr Ala Pro Arg Tyr Gly1295 1300
1305Ile Arg Gly Ile Pro Thr Leu Leu Leu Phe Arg Asn Gly Glu His1310
1315 1320His His His His His His His His
His Val1325 1330402721DNAArtificialtruncated lac Z gene
sequence 40cctaggatga tagatcccgt cgttttacaa cgtcgtgact gggaaaaccc
tggcgttacc 60caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
cgaagaggcc 120cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg
ctttgcctgg 180tttccggtac cagaagcggt gccggaaagc tggctggagt gcgatcttcc
tgaggccgat 240actgtcgtcg tcccctcaaa ctggcagatg cacggttacg atgcgcccat
ctacaccaac 300gtaacctatc ccattacggt caatccgccg tttgttccca cggagaatcc
gacgggttgt 360tactcgctca catttaatgt tgatgaaagc tggctacagg aaggccagac
gcgaattatt 420tttgatggcg ttaactcggc gtttcatctg tggtgcaacg ggcgctgggt
cggttacggc 480caggacagtc gtttgccgtc tgaatttgac ctgagcgcat ttttacgcgc
cggagaaaac 540cgcctcgcgg tgatggtgct gcgttggagt gacggcagtt atctggaaga
tcaggatatg 600tggcggatga gcggcatttt ccgtgacgtc tcgttgctgc ataaaccgac
tacacaaatc 660agcgatttcc atgttgccac tcgctttaat gatgatttca gccgcgctgt
actggaggct 720gaagttcaga tgtgcggcga gttgcgtgac tacctacggg taacagtttc
tttatggcag 780ggtgaaacgc aggtcgccag cggcaccgcg cctttcggcg gtgaaattat
cgatgagcgt 840ggtggttatg ccgatcgcgt cacactacgt ctgaacgtcg aaaacccgaa
actgtggagc 900gccgaaatcc cgaatctcta tcgtgcggtg gttgaactgc acaccgccga
cggcacgctg 960attgaagcag aagcctgcga tgtcggtttc cgcgaggtgc ggattgaaaa
tggtctgctg 1020ctgctgaacg gcaagccgtt gctgattcga ggcgttaacc gtcacgagca
tcatcctctg 1080catggtcagg tcatggatga gcagacgatg gtgcaggata tcctgctgat
gaagcagaac 1140aactttaacg ccgtgcgctg ttcgcattat ccgaaccatc cgctgtggta
cacgctgtgc 1200gaccgctacg gcctgtatgt ggtggatgaa gccaatattg aaacccacgg
catggtgcca 1260atgaatcgtc tgaccgatga tccgcgctgg ctaccggcga tgagcgaacg
cgtaacgcga 1320atggtgcagc gcgatcgtaa tcacccgagt gtgatcatct ggtcgctggg
gaatgaatca 1380ggccacggcg ctaatcacga cgcgctgtat cgctggatca aatctgtcga
tccttcccgc 1440ccggtgcagt atgaaggcgg cggagccgac accacggcca ccgatattat
ttgcccgatg 1500tacgcgcgcg tggatgaaga ccagcccttc ccggctgtgc cgaaatggtc
catcaaaaaa 1560tggctttcgc tacctggaga gacgcgcccg ctgatccttt gcgaatacgc
ccacgcgatg 1620ggtaacagtc ttggcggttt cgctaaatac tggcaggcgt ttcgtcagta
tccccgttta 1680cagggcggct tcgtctggga ctgggtggat cagtcgctga ttaaatatga
tgaaaacggc 1740aacccgtggt cggcttacgg cggtgatttt ggcgatacgc cgaacgatcg
ccagttctgt 1800atgaacggtc tggtctttgc cgaccgcacg ccgcatccag cgctgacgga
agcaaaacac 1860cagcagcagt ttttccagtt ccgtttatcc gggcaaacca tcgaagtgac
cagcgaatac 1920ctgttccgtc atagcgataa cgagctcctg cactggatgg tggcgctgga
tggtaagccg 1980ctggcaagcg gtgaagtgcc tctggatgtc gctccacaag gtaaacagtt
gattgaactg 2040cctgaactac cgcagccgga gagcgccggg caactctggc tcacagtacg
cgtagtgcaa 2100ccgaacgcga ccgcatggtc agaagccggg cacatcagcg cctggcagca
gtggcgtctg 2160gcggaaaacc tcagtgtgac gctccccgcc gcgtcccacg ccatcccgca
tctgaccacc 2220agcgaaatgg atttttgcat cgagctgggt aataagcgtt ggcaatttaa
ccgccagtca 2280ggctttcttt cacagatgtg gattggcgat aaaaaacaac tgctgacgcc
gctgcgcgat 2340cagttcaccc gtgcaccgct ggataacgac attggcgtaa gtgaagcgac
ccgcattgac 2400cctaacgcct gggtcgaacg ctggaaggcg gcgggccatt accaggccga
agcagcgttg 2460ttgcagtgca cggcagatac acttgctgat gcggtgctga ttacgaccgc
tcacgcgtgg 2520cagcatcagg ggaaaacctt atttatcagc cggaaaacct accggattga
tggtagtggt 2580caaatggcga ttaccgttga tgttgaagtg gcgagcgata caccgcatcc
ggcgcggatt 2640ggcctgaact gccagctggc gcaggtagca gagcgggtaa actggctcgg
attagggccg 2700caagaaaact atccccctag g
2721412238PRTArtificialpTRC 260 kDa expression product 41Met
His Gly Ser Met Cys Asp Arg Ile Ile His Leu Thr Asp Asp Cys1
5 10 15Phe Asp Thr Asp Val Leu Arg
Ala Asp Gly Ala Arg Leu Val Asp Phe20 25
30Trp Ala Glu Trp Cys Gly Pro Arg Met Cys Ile Ala Pro Ile Leu Asp35
40 45Glu Arg Ala Asp Glu Tyr Gln Gly Arg Leu
Thr Val Ala Arg Leu Asn50 55 60Ile Asp
Gln Asn Pro Gly Thr Ala Pro Arg Tyr Gly Ile Arg Gly Ile65
70 75 80Pro Thr Leu Leu Leu Phe Arg
Asn Gly Glu Gly Thr Met Cys Asp Arg85 90
95Ile Ile His Leu Thr Asp Asp Cys Phe Asp Thr Asp Val Leu Arg Ala100
105 110Asp Gly Ala Arg Leu Val Asp Phe Trp
Ala Glu Trp Cys Gly Pro Arg115 120 125Met
Cys Ile Ala Pro Ile Leu Asp Glu Arg Ala Asp Glu Tyr Gln Gly130
135 140Arg Leu Thr Val Ala Arg Leu Asn Ile Asp Gln
Asn Pro Gly Thr Ala145 150 155
160Pro Arg Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu Leu Phe Arg
Asn165 170 175Gly Glu Glu Phe Met Cys Asp
Arg Ile Ile His Leu Thr Asp Asp Cys180 185
190Phe Asp Thr Asp Val Leu Arg Ala Asp Gly Ala Arg Leu Val Asp Phe195
200 205Trp Ala Glu Trp Cys Gly Pro Arg Met
Cys Ile Ala Pro Ile Leu Asp210 215 220Glu
Arg Ala Asp Glu Tyr Gln Gly Arg Leu Thr Val Ala Arg Leu Asn225
230 235 240Ile Asp Gln Asn Pro Gly
Thr Ala Pro Arg Tyr Gly Ile Arg Gly Ile245 250
255Pro Thr Leu Leu Leu Phe Arg Asn Gly Glu Thr Gly Met Cys Asp
Arg260 265 270Ile Ile His Leu Thr Asp Asp
Cys Phe Asp Thr Asp Val Leu Arg Ala275 280
285Asp Gly Ala Arg Leu Val Asp Phe Trp Ala Glu Trp Cys Gly Pro Arg290
295 300Met Cys Ile Ala Pro Ile Leu Asp Glu
Arg Ala Asp Glu Tyr Gln Gly305 310 315
320Arg Leu Thr Val Ala Arg Leu Asn Ile Asp Gln Asn Pro Gly
Thr Ala325 330 335Pro Arg Tyr Gly Ile Arg
Gly Ile Pro Thr Leu Leu Leu Phe Arg Asn340 345
350Gly Glu Ala Gly Met Cys Asp Arg Ile Ile His Leu Thr Asp Asp
Cys355 360 365Phe Asp Thr Asp Val Leu Arg
Ala Asp Gly Ala Ile Leu Val Asp Phe370 375
380Trp Ala Glu Trp Cys Gly Pro Arg Met Cys Ile Ala Pro Ile Leu Asp385
390 395 400Glu Ile Ala Asp
Glu Tyr Gln Gly Arg Leu Thr Val Ala Arg Leu Asn405 410
415Ile Asp Gln Asn Pro Gly Thr Ala Pro Arg Tyr Gly Ile Arg
Gly Ile420 425 430Pro Thr Leu Leu Leu Phe
Arg Asn Gly Glu Leu Glu Met Cys Asp Arg435 440
445Ile Ile His Leu Thr Asp Asp Cys Phe Asp Thr Asp Val Leu Arg
Ala450 455 460Asp Gly Ala Arg Leu Val Asp
Phe Trp Ala Glu Trp Cys Gly Pro Arg465 470
475 480Met Cys Ile Ala Pro Ile Leu Asp Glu Arg Ala Asp
Glu Tyr Gln Gly485 490 495Arg Leu Thr Val
Ala Arg Leu Asn Ile Asp Gln Asn Pro Gly Thr Ala500 505
510Pro Arg Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu Leu Phe
Arg Asn515 520 525Gly Glu Gly Thr Met Cys
Asp Arg Ile Ile His Leu Thr Asp Asp Cys530 535
540Phe Asp Thr Asp Val Leu Arg Ala Asp Gly Ala Arg Leu Val Asp
Phe545 550 555 560Trp Ala
Glu Trp Cys Gly Pro Arg Met Cys Ile Ala Pro Ile Leu Asp565
570 575Glu Arg Ala Asp Glu Tyr Gln Gly Arg Leu Thr Val
Ala Arg Leu Asn580 585 590Ile Asp Gln Asn
Pro Gly Thr Ala Pro Arg Tyr Gly Ile Arg Gly Ile595 600
605Pro Thr Leu Leu Leu Phe Arg Asn Gly Glu Glu Phe Met Cys
Asp Arg610 615 620Ile Ile His Leu Thr Asp
Asp Cys Phe Asp Thr Asp Val Leu Arg Ala625 630
635 640Asp Gly Ala Arg Leu Val Asp Phe Trp Ala Glu
Trp Cys Gly Pro Arg645 650 655Met Cys Ile
Ala Pro Ile Leu Asp Glu Arg Ala Asp Glu Tyr Gln Gly660
665 670Arg Leu Thr Val Ala Arg Leu Asn Ile Asp Gln Asn
Pro Gly Thr Ala675 680 685Pro Arg Tyr Gly
Ile Arg Gly Ile Pro Thr Leu Leu Leu Phe Arg Asn690 695
700Gly Glu Thr Gly Met Cys Asp Arg Ile Ile His Leu Thr Asp
Asp Cys705 710 715 720Phe
Asp Thr Asp Val Leu Arg Ala Asp Gly Ala Arg Leu Val Asp Phe725
730 735Trp Ala Glu Trp Cys Gly Pro Arg Met Cys Ile
Ala Pro Ile Leu Asp740 745 750Glu Arg Ala
Asp Glu Tyr Gln Gly Arg Leu Thr Val Ala Arg Leu Asn755
760 765Ile Asp Gln Asn Pro Gly Thr Ala Pro Arg Tyr Gly
Ile Arg Gly Ile770 775 780Pro Thr Leu Leu
Leu Phe Arg Asn Gly Glu Ala Gly Met Cys Asp Arg785 790
795 800Ile Ile His Leu Thr Asp Asp Cys Phe
Asp Thr Asp Val Leu Arg Ala805 810 815Asp
Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp Cys Gly Pro Arg820
825 830Met Cys Ile Ala Pro Ile Leu Asp Glu Ile Ala
Asp Glu Tyr Gln Gly835 840 845Arg Leu Thr
Val Ala Arg Leu Asn Ile Asp Gln Asn Pro Gly Thr Ala850
855 860Pro Arg Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu
Leu Phe Arg Asn865 870 875
880Gly Glu Pro Arg Met Ile Asp Pro Val Val Leu Gln Arg Arg Asp Trp885
890 895Glu Asn Pro Gly Val Thr Gln Leu Asn
Arg Leu Ala Ala His Pro Pro900 905 910Phe
Ala Ser Trp Arg Asn Ser Glu Glu Ala Arg Thr Asp Arg Pro Ser915
920 925Gln Gln Leu Arg Ser Leu Asn Gly Glu Trp Arg
Phe Ala Trp Phe Pro930 935 940Val Pro Glu
Ala Val Pro Glu Ser Trp Leu Glu Cys Asp Leu Pro Glu945
950 955 960Ala Asp Thr Val Val Val Pro
Ser Asn Trp Gln Met His Gly Tyr Asp965 970
975Ala Pro Ile Tyr Thr Asn Val Thr Tyr Pro Ile Thr Val Asn Pro Pro980
985 990Phe Val Pro Thr Glu Asn Pro Thr Gly
Cys Tyr Ser Leu Thr Phe Asn995 1000
1005Val Asp Glu Ser Trp Leu Gln Glu Gly Gln Thr Arg Ile Ile Phe1010
1015 1020Asp Gly Val Asn Ser Ala Phe His
Leu Trp Cys Asn Gly Arg Trp1025 1030
1035Val Gly Tyr Gly Gln Asp Ser Arg Leu Pro Ser Glu Phe Asp Leu1040
1045 1050Ser Ala Phe Leu Arg Ala Gly Glu
Asn Arg Leu Ala Val Met Val1055 1060
1065Leu Arg Trp Ser Asp Gly Ser Tyr Leu Glu Asp Gln Asp Met Trp1070
1075 1080Arg Met Ser Gly Ile Phe Arg Asp
Val Ser Leu Leu His Lys Pro1085 1090
1095Thr Thr Gln Ile Ser Asp Phe His Val Ala Thr Arg Phe Asn Asp1100
1105 1110Asp Phe Ser Arg Ala Val Leu Glu
Ala Glu Val Gln Met Cys Gly1115 1120
1125Glu Leu Arg Asp Tyr Leu Arg Val Thr Val Ser Leu Trp Gln Gly1130
1135 1140Glu Thr Gln Val Ala Ser Gly Thr
Ala Pro Phe Gly Gly Glu Ile1145 1150
1155Ile Asp Glu Arg Gly Gly Tyr Ala Asp Arg Val Thr Leu Arg Leu1160
1165 1170Asn Val Glu Asn Pro Lys Leu Trp
Ser Ala Glu Ile Pro Asn Leu1175 1180
1185Tyr Arg Ala Val Val Glu Leu His Thr Ala Asp Gly Thr Leu Ile1190
1195 1200Glu Ala Glu Ala Cys Asp Val Gly
Phe Arg Glu Val Arg Ile Glu1205 1210
1215Asn Gly Leu Leu Leu Leu Asn Gly Lys Pro Leu Leu Ile Arg Gly1220
1225 1230Val Asn Arg His Glu His His Pro
Leu His Gly Gln Val Met Asp1235 1240
1245Glu Gln Thr Met Val Gln Asp Ile Leu Leu Met Lys Gln Asn Asn1250
1255 1260Phe Asn Ala Val Arg Cys Ser His
Tyr Pro Asn His Pro Leu Trp1265 1270
1275Tyr Thr Leu Cys Asp Arg Tyr Gly Leu Tyr Val Val Asp Glu Ala1280
1285 1290Asn Ile Glu Thr His Gly Met Val
Pro Met Asn Arg Leu Thr Asp1295 1300
1305Asp Pro Arg Trp Leu Pro Ala Met Ser Glu Arg Val Thr Arg Met1310
1315 1320Val Gln Arg Asp Arg Asn His Pro
Ser Val Ile Ile Trp Ser Leu1325 1330
1335Gly Asn Glu Ser Gly His Gly Ala Asn His Asp Ala Leu Tyr Arg1340
1345 1350Trp Ile Lys Ser Val Asp Pro Ser
Arg Pro Val Gln Tyr Glu Gly1355 1360
1365Gly Gly Ala Asp Thr Thr Ala Thr Asp Ile Ile Cys Pro Met Tyr1370
1375 1380Ala Arg Val Asp Glu Asp Gln Pro
Phe Pro Ala Val Pro Lys Trp1385 1390
1395Ser Ile Lys Lys Trp Leu Ser Leu Pro Gly Glu Thr Arg Pro Leu1400
1405 1410Ile Leu Cys Glu Tyr Ala His Ala
Met Gly Asn Ser Leu Gly Gly1415 1420
1425Phe Ala Lys Tyr Trp Gln Ala Phe Arg Gln Tyr Pro Arg Leu Gln1430
1435 1440Gly Gly Phe Val Trp Asp Trp Val
Asp Gln Ser Leu Ile Lys Tyr1445 1450
1455Asp Glu Asn Gly Asn Pro Trp Ser Ala Tyr Gly Gly Asp Phe Gly1460
1465 1470Asp Thr Pro Asn Asp Arg Gln Phe
Cys Met Asn Gly Leu Val Phe1475 1480
1485Ala Asp Arg Thr Pro His Pro Ala Leu Thr Glu Ala Lys His Gln1490
1495 1500Gln Gln Phe Phe Gln Phe Arg Leu
Ser Gly Gln Thr Ile Glu Val1505 1510
1515Thr Ser Glu Tyr Leu Phe Arg His Ser Asp Asn Glu Leu Leu His1520
1525 1530Trp Met Val Ala Leu Asp Gly Lys
Pro Leu Ala Ser Gly Glu Val1535 1540
1545Pro Leu Asp Val Ala Pro Gln Gly Lys Gln Leu Ile Glu Leu Pro1550
1555 1560Glu Leu Pro Gln Pro Glu Ser Ala
Gly Gln Leu Trp Leu Thr Val1565 1570
1575Arg Val Val Gln Pro Asn Ala Thr Ala Trp Ser Glu Ala Gly His1580
1585 1590Ile Ser Ala Trp Gln Gln Trp Arg
Leu Ala Glu Asn Leu Ser Val1595 1600
1605Thr Leu Pro Ala Ala Ser His Ala Ile Pro His Leu Thr Thr Ser1610
1615 1620Glu Met Asp Phe Cys Ile Glu Leu
Gly Asn Lys Arg Trp Gln Phe1625 1630
1635Asn Arg Gln Ser Gly Phe Leu Ser Gln Met Trp Ile Gly Asp Lys1640
1645 1650Lys Gln Leu Leu Thr Pro Leu Arg
Asp Gln Phe Thr Arg Ala Pro1655 1660
1665Leu Asp Asn Asp Ile Gly Val Ser Glu Ala Thr Arg Ile Asp Pro1670
1675 1680Asn Ala Trp Val Glu Arg Trp Lys
Ala Ala Gly His Tyr Gln Ala1685 1690
1695Glu Ala Ala Leu Leu Gln Cys Thr Ala Asp Thr Leu Ala Asp Ala1700
1705 1710Val Leu Ile Thr Thr Ala His Ala
Trp Gln His Gln Gly Lys Thr1715 1720
1725Leu Phe Ile Ser Arg Lys Thr Tyr Arg Ile Asp Gly Ser Gly Gln1730
1735 1740Met Ala Ile Thr Val Asp Val Glu
Val Ala Ser Asp Thr Pro His1745 1750
1755Pro Ala Arg Ile Gly Leu Asn Cys Gln Leu Ala Gln Val Ala Glu1760
1765 1770Arg Val Asn Trp Leu Gly Leu Gly
Pro Gln Glu Asn Tyr Pro Pro1775 1780
1785Arg Met Cys Asp Arg Ile Ile His Leu Thr Asp Asp Cys Phe Asp1790
1795 1800Thr Asp Val Leu Arg Ala Asp Gly
Ala Arg Leu Val Asp Phe Trp1805 1810
1815Ala Glu Trp Cys Gly Pro Arg Met Cys Ile Ala Pro Ile Leu Asp1820
1825 1830Glu Arg Ala Asp Glu Tyr Gln Gly
Arg Leu Thr Val Ala Arg Leu1835 1840
1845Asn Ile Asp Gln Asn Pro Gly Thr Ala Pro Arg Tyr Gly Ile Arg1850
1855 1860Gly Ile Pro Thr Leu Leu Leu Phe
Arg Asn Gly Glu Gly Thr Met1865 1870
1875Cys Asp Arg Ile Ile His Leu Thr Asp Asp Cys Phe Asp Thr Asp1880
1885 1890Val Leu Arg Ala Asp Gly Ala Arg
Leu Val Asp Phe Trp Ala Glu1895 1900
1905Trp Cys Gly Pro Arg Met Cys Ile Ala Pro Ile Leu Asp Glu Arg1910
1915 1920Ala Asp Glu Tyr Gln Gly Arg Leu
Thr Val Ala Arg Leu Asn Ile1925 1930
1935Asp Gln Asn Pro Gly Thr Ala Pro Arg Tyr Gly Ile Arg Gly Ile1940
1945 1950Pro Thr Leu Leu Leu Phe Arg Asn
Gly Glu Glu Phe Met Cys Asp1955 1960
1965Arg Ile Ile His Leu Thr Asp Asp Cys Phe Asp Thr Asp Val Leu1970
1975 1980Arg Ala Asp Gly Ala Arg Leu Val
Asp Phe Trp Ala Glu Trp Cys1985 1990
1995Gly Pro Arg Met Cys Ile Ala Pro Ile Leu Asp Glu Arg Ala Asp2000
2005 2010Glu Tyr Gln Gly Arg Leu Thr Val
Ala Arg Leu Asn Ile Asp Gln2015 2020
2025Asn Pro Gly Thr Ala Pro Arg Tyr Gly Ile Arg Gly Ile Pro Thr2030
2035 2040Leu Leu Leu Phe Arg Asn Gly Glu
Thr Gly Met Cys Asp Arg Ile2045 2050
2055Ile His Leu Thr Asp Asp Cys Phe Asp Thr Asp Val Leu Arg Ala2060
2065 2070Asp Gly Ala Arg Leu Val Asp Phe
Trp Ala Glu Trp Cys Gly Pro2075 2080
2085Arg Met Cys Ile Ala Pro Ile Leu Asp Glu Arg Ala Asp Glu Tyr2090
2095 2100Gln Gly Arg Leu Thr Val Ala Arg
Leu Asn Ile Asp Gln Asn Pro2105 2110
2115Gly Thr Ala Pro Arg Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu2120
2125 2130Leu Phe Arg Asn Gly Glu Ala Gly
Met Cys Asp Arg Ile Ile His2135 2140
2145Leu Thr Asp Asp Cys Phe Asp Thr Asp Val Leu Arg Ala Asp Gly2150
2155 2160Ala Ile Leu Val Asp Phe Trp Ala
Glu Trp Cys Gly Pro Arg Met2165 2170
2175Cys Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp Glu Tyr Gln Gly2180
2185 2190Arg Leu Thr Val Ala Arg Leu Asn
Ile Asp Gln Asn Pro Gly Thr2195 2200
2205Ala Pro Arg Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu Leu Phe2210
2215 2220Arg Asn Gly Glu His His His His
His His His His His His Val2225 2230
2235
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