Patent application title: SOLUBILIZATION AND PURIFICATION OF A TARGET PROTEIN FUSED TO A MUTANT MALTOSE-BINDING PROTEIN
Paul Riggs (Hooksett, NH, US)
Pei-Chung Hsieh (Topsfield, MA, US)
Iris Walker (Rowley, MA, US)
Paul A. Colussi (Gloucester, MA, US)
Paul A. Colussi (Gloucester, MA, US)
Mehul Ganatra (Gloucester, MA, US)
Christopher H. Taron (Essex, MA, US)
Christopher H. Taron (Essex, MA, US)
NEW ENGLAND BIOLABS, INC.
IPC8 Class: AC12N121FI
Class name: Bacteria or actinomycetales; media therefor transformants (e.g., recombinant dna or vector or foreign or exogenous gene containing, fused bacteria, etc.) escherichia (e.g., e. coli, etc.)
Publication date: 2011-01-27
Patent application number: 20110020911
Patent application title: SOLUBILIZATION AND PURIFICATION OF A TARGET PROTEIN FUSED TO A MUTANT MALTOSE-BINDING PROTEIN
Christopher H. Taron
Paul A. Colussi
HARRIET M. STRIMPEL, D. Phil.
Origin: IPSWICH, MA US
IPC8 Class: AC12N121FI
Publication date: 01/27/2011
Patent application number: 20110020911
Methods and compositions are provided for increasing at least one of: (i)
binding affinity of a target protein for a maltodextrin substrate and/or
(ii) solubility of a target protein. The methods and compositions relate
to a modified maltose-binding protein.
7. A vector comprising a nucleic acid sequence encoding a protein having a sequence corresponding to SEQ ID NO: 2 wherein residue S146 and/or A313 is mutated.
8. A vector comprising a nucleic acid encoding a protein comprising SEQ ID NO:4 or SEQ ID NO: 6.
9. A vector comprising a nucleic acid encoding a protein according to claim 7 or 8 and fused to a target protein.
10. A host cell containing a vector according to claim 7.
11. A host cell containing a vector according to claim 8.
12. A host cell containing a vector according to claim 9.
13. A method for expressing a protein with increased solubility, comprising:(a) Introducing a vector according to claim 9 into a host cell; and(b) Expressing the fusion protein with increased solubility compared to the target protein in the absence of a variant MBP.
This application is a divisional of U.S. application Ser. No. 12/297,105 filed Oct. 14, 2008, which is a §371 application of international application number PCT/US07/09100 filed Apr. 14, 2007, which claims priority from U.S. provisional application No. 60/792,133 filed Apr. 14, 2006, herein incorporated by reference.
Recombinant proteins have many uses in biotechnology, whenever large amounts of pure protein are needed. Microbial expression systems such as Escherichia coil (E. coli) and yeast are often the first choice due to their low cost and high yield. When expressing foreign proteins in E. coli, it is not uncommon to encounter problems of low levels of expression and/or insolubility of the protein. Even if the protein is expressed well and remains soluble, it must be purified from the myriad of other proteins in the cell extract. To facilitate the expression and purification of a target protein, one method that is in common use is to fuse an affinity tag to the protein. A good affinity tag has properties that facilitate high-level expression when fused to the N-terminus of the target protein, as well as providing a simple one-step affinity purification that allows the target protein to be purified from the expression mileu.
The maltose-binding protein (MBP) of E. coli is commonly used as an affinity tag for expression and purification of foreign proteins produced in E. coli. The natural role of MBP is to bind maltodextrins at the outer membrane porin and release them to the MaIEFK transport apparatus in the inner membrane. Fusion of the C-terminus of MBP to the N-terminus of a target protein permits the expression of the fusion protein in E. coli (FIG. 1). MBP and MBP fusions can be purified in a single step by binding to a chromatography matrix containing any of a number of glucose-α1→4-glucose polysaccharides such as amylose, starch or other maltodextrins (U.S. Pat. No. 5,643,758). Many proteins that are soluble in their native host are insoluble when expressed as a recombinant protein. For many of these proteins, fusion to MBP renders them soluble (Kapust & Waugh, Protein Sci. 8:1668-74 (1999)).
The utility of MBP as an affinity tag is tempered by the fact that depending on the protein in a MBP-target protein purification, some fusions don't bind to the affinity matrix as well as others. In addition, the presence of non-ionic detergents such as Triton X100 and Tween 20 can interfere with binding, especially for MBP-target protein fusions that have an inherently lower affinity.
Researchers have used the structure of MBP to make directed mutations in order to alter the binding properties of MBP. The X-ray crystal structure of MBP has been reported by Spurlino et al., J. Biol. Chem. 266:5202-5219 (1991). MBP consists of two domains, with a cleft between the domains where the polysaccharide binds. The domain that contains the N-terminus is named the N domain, and the domain that contains the C-terminus is named the C domain. Three loops cross between the two domains to form a hinge. Two groups have used the structure to make directed mutations to the region behind the hinges that increase the affinity of MBP for maltose and maltotriose (Marvin et al., Nature Structural Biology 8:795-798 (2001); Telmer & Shilton, Journal of Biol. Chem. 278:34555-34567 (2003)). However, this approach has an inherent disadvantage, since the modifications to MBP increase the hydrophobicity of the surface of the protein and thus decrease its solubility. This reduces its utility as an affinity tag by increasing its tendency to render a fusion protein insoluble.
In an embodiment of the invention, a modified MBP fusion protein is provided that is characterized by an MBP amino acid sequence having a mutation wherein the mutation causes the modified MBP fusion protein to have at least one property selected from (i) an increased affinity for a maltodextrin substrate and (ii) an increased solubility when fused to a target protein having limited solubility. In a further embodiment of the invention, the modified MBP has a mutation located in the hinge region between helices XI and XII or in a region within 10 Å of A313 of the protein. For example, the mutation may be located in the C domain or in a region within 10 Å of S146, at the beginning of β-sheet F of the protein. The modification may be specifically A313V or S146T.
In a further embodiment of the invention, the modified MBP may be fused to a target protein to form a fusion protein and may, for example, have a solubility that is greater than the solubility of an unmodified MBP protein fused to the target protein.
In an embodiment of the invention, the modified MBP may have an amino sequence selected from SEQ ID NO:4 or SEQ ID NO:6 or a DNA sequence encoding the protein selected from SEQ ID NO:3 or SEQ ID NO:5. The DNA may be incorporated into a vector such as a pKLAC1 vector for expression in a host cell such as a Kluyveromyces or E. coli.
In an embodiment of the invention, a method is provided for purifying a protein that includes: expressing in a host cell such as a Kluyveromyces or E. coli, a fusion protein that includes any of the modified MBPs described above and a target protein. The modified MBP fusion protein is permitted to bind to a matrix such as a polysaccharide exemplified by a maltodextrin. The fusion protein can then be eluted from the matrix in a selected buffer to obtain the purified protein.
In a further embodiment of the invention, a method is provided for solubilizing a target protein that includes expressing an MBP mutant fused to a target protein so that the fusion protein is solubilized to an extent greater than can be observed in the absence of the mutant MBP and to an extent greater than observed using a non-mutated MBP. Examples of mutations include a A313V mutation and/or an S146T mutation.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a schematic describing the cloning and purification of a target protein by expressing a DNA encoding an MBP fused to a target protein, allowing the fusion protein to selectively bind to amylose, eluting the target protein in a maltose-containing buffer and then recovering the target protein from the purified fusion protein by protease cleavage.
FIG. 2 provides sequences comparing wild-type MBPs with modified MBPs.
FIG. 2A: The DNA sequence (SEQ ID NO:1) encoding wild-type MBP (SEQ ID NO:2) from pMAL-c2X.
FIG. 2B: The DNA sequence (SEQ ID NO:3) encoding the MBP mutant A9 (SEQ ID NO:4). Changes in the modified MBP sequences are indicated in bold.
FIG. 2C: The DNA sequence (SEQ ID NO:5) encoding the MBP mutant G8-1 (SEQ ID NO:6). Changes in the modified MBP DNA and amino acid sequences are indicated in bold.
FIG. 3 shows a histogram comparing yields of protein using wild-type MBP (pKO1483), or modified MBPs (A9 or G8-1). The yields of MBPs are provided in mg/500 mls of culture.
FIG. 4 shows on an SDS-PAGE gel the purified product of MBP-Klenow from pMAL containing wild-type MBP, MBP A313V and S146T modifications. Plasmids: WT=pIH1062, A313V=pIH1643, S136T=pIH1644. Lanes: CE=crude extract, FT=flow though, and E=eluate. Equal portions of the indicated fraction were loaded in each lane. A significantly higher yield of MBP fusion protein was obtained from A313V mutant MBP than from wild-type MBP (also see Table 1).
FIG. 5. Sequence of pMB50 (SEQ ID NO:7) used for expressing MBP-CBD fusion protein (see Table 2).
FIG. 6. SDS-PAGE gels showing the effect on solubility of fusing wild-type and modified MBPs to DHFR and GAPDH. DHFR fusion plasmids: WT=pIH1616; A313V=pIH1617; S146T=pIH1618; DM=pIH1646. GAPDH fusion plasmids: WT=pIH1625; A313V=pIH1626; S146T=pIH1627; DM=pIH1645. Lanes: T=total cell extract; S=soluble extract; I=resuspension of insoluble material. Equal portions of the indicated fraction were loaded in each lane. The ratio of soluble to insoluble protein was greater for both mutants compared with wild-type MBPs.
FIG. 7 provides the sequence of pKLMF2-PMΔSal (SEQ ID NO:8).
FIG. 8 shows fractions from the affinity purification, using an amylose resin column, of a fusion protein on an SDS-PAGE gel. An enhanced yield of modified MBP fusion protein was produced by expressing MBP (A313V)PMΔSal in Kluyveromyces lactis cells carrying an MBP (A313v)-PMΔSal expression cassette.
Lane 1--NEB Broad range markers, Ipswich, Mass.;
Lane 2--crude extract load;
Lane 3--column flow-through;
Lane 4--column wash;
Lanes 5, 6 and 7, elution with column buffer+maltose.
DETAILED DESCRIPTION OF THE EMBODIMENTS
Terms that are used herein are discussed below.
"Wild-type" MBP includes the MBP protein produced by expression from a derivative of one of the pMAL-2 plasmids that has a stop codon in the polylinker, for example pKO1483.
"Enhanced solubility of a protein fused to a mutant MBP" is an increase in the amount of soluble protein when compared to that same protein fused to wild-type MBP. Solubility can be expressed as the ratio of soluble protein to the total amount of that protein present before insoluble material is removed, for example by centrifugation.
"Increased affinity of a mutant MBP" or "mutant MBP fusion protein" includes: an increase in the amount of protein that binds to a solid substrate such as a maltodextrin under a defined set of conditions. The efficacy of the affinity purification can be expressed as the ratio of protein that binds to maltodextrin under the specified conditions and is then eluted with a specified buffer to the total amount of that protein applied to the column.
The present embodiments of the invention provide MBP mutants which when fused to a target protein enhance the solubility of the fusion protein during expression in vivo and can also improve the affinity of the fusion protein during purification. The present embodiments include mutant MBPs that show increased binding to a polysaccharide media, such as one that includes a maltodextrin, when applied to the media under conditions where wild-type MBP shows partial binding. The modified MBPs are then eluted from the media using a solution of, for example, a soluble maltodextrin, yielding at least 1.5 to 10-fold more protein when compared to wild-type MBP.
In order to discover these improved mutants of MBP, technical hurdles had to be overcome which include developing techniques which enable a large number of samples to be handled. This required improved methods for breaking up host cells to release solubilized fusion protein where sonication is not practical for large scale purification and lysis buffers could interfere with affinity binding of MBP. It was discovered that by titrating the detergent and the lysozyme, it was possible to identify the appropriate concentration and ratio of these lysis reagents to effectively break up host cells without negatively impacting binding affinity.
In order to screen for mutants with desired binding affinity properties, 96 well microplates were used where each well contained a micro matrix for binding fusion protein and a filter apparatus removed contaminating materials in the filtrate. This made possible rapid screening of large numbers of samples.
The screening methods for obtaining and testing modified mutant MBP proteins as improved tags for purifying proteins are described in the examples. Such modified MBPs that have a higher affinity for a matrix solve the problem associated with wild-type MBP of MBP fusions proteins that bind poorly to a matrix or where binding is disrupted by the presence of non-ionic detergent.
In the examples, two mutations (S146T and A313V) are described with the desired properties of improved solubility and improved binding affinity to a polysaccharide matrix that were isolated using the screening procedure. The S146T mutation is in the C domain of MBP at the beginning of β-sheet F. When a target protein that is insoluble is fused to MBP containing this mutation, the solubility of the fusion protein is enhanced. The A313V mutation described herein is located in the third hinge region that crosses between the two domains, specifically, in the loop between helices XI and XII. This mutation enhances both the solubility and the affinity of fusion proteins. When expressing foreign proteins in E. coli, the protein may be partially or completely expressed in the form of insoluble aggregates. In particular examples, solubility may be increased by 1.1 or greater upwards with an upper limit of total solubility.
All references cited herein, as well as U.S. application Ser. No. 12/297,105 filed Oct. 14, 2008 and U.S. provisional application No. 60/792,133 filed Apr. 14, 2006, are incorporated by reference.
Restriction enzymes, β-agarase, DNA polymerases, T4 ligase, Antarctic phosphatase, Litmus 38, the pMAL Protein Fusion and Purification System including pMAL-c2X and pMAL-c2G, amylose resin (#E8021), anti-MBP monoclonal antibody linked to horse radish peroxidase (#E8038), the USER Friendly Cloning kit, the K. lactis Protein Expression Kit including the vector pKLAC1, host strains TB1, ER1992, ER2502, ER2984, NEB 5-alpha, and NEB Turbo, and synthetic oligonucleotides were obtained from New England Biolabs, Inc. (NEB), Ipswich, Mass. Unifilter 800 microtiter microplates with filter bottoms were purchased from Whatman, Brentford, England. The Minelute DNA Extraction and Qiaprep Spin kits were purchased from Qiagen, Valencia, Calif. Mega 10 was purchased from Dojindo, Gaithersburg, Md. Hen egg white lysozyme, Coomassie brilliant blue R and acid washed glass beads (425-600 micron) were purchased from Sigma-Aldrich, St. Louis, Mo. Sea Plaque GTG low melting temperature agarose was purchased from Cambrex, E. Rutherford, N.J. Disposable polypropylene columns (#732-6008) were purchased from BioRad, Hercules, Calif. 10-20% gradient gels were purchased from either Daiichi, Tokyo, Japan or InVitrogen/Novex, Carlsbad, Calif. The Complete® protease inhibitor cocktail was purchased from Roche, Basel, Switzerland. SimplyBlue Safestain was purchased from Invitrogen, Carlsbad, Calif. The human dihydrofolate reductase (DHFR) cDNA clone pOTB7-DHFR was purchased from Invitrogen (MGC:857). The GAPDH gene was obtained from pJF931 (Fox et al. FEBS Lett. 537:53-57 (2003).
The Serracia marscesens nuclease was obtained as described in PCT/US05/28739. Minipreps of plasmid DNA were prepared using the Qiaprep Spin kit. Random PCR mutagenesis was carried out as described in Fromant et al. Analytical Biochemistry 224, 347-353 (1995). PCR was carried out using Vent® DNA polymerase except as noted. DNA fragments were gel-purified by electrophoresis on 1% Sea Plaque GTG low melting temperature agarose, cutting out the band, and either purifying the DNA using the Minelute DNA Extraction kit, or melting it at 75° C. for 5 minutes, cooling to 37° C., and digesting with β-agarOse for 1-2 h. DNA sequencing was performed on Applied Biosystems's (ABI's) automated DNA Sequencer model 3100 ABI, using Big Dye labeled dye-terminator chemistry (ABI, Foster City, Calif.). SDS-PAGE was carried out according to the instructions of the acrylamide gel provider, and proteins were visualized by staining with Coomassie brilliant blue R except where noted otherewise.
MBP was expressed from either pMal-c2X or pMal-c2G or a derivative of pMal-c2G. The numbering of bases to identify mutations in malE refers to the base number in the pMAL-c2X sequence ((FIGS. 2A-1, 2A-2, 2B-1, 2B-2, 2C-1 and 2C-2 (SEQ ID NOS:1-6)). The pMAL-c2G derivative pSN1578 was created by cleaving the plasmid with BsmI and BsiWI, treating the product with DNA polymerase Klenow fragment plus all four dNTPs, followed by ligation to create a deletion within the malE gene.
Site-directed mutagenesis was carried out using a four primer PCR mutagenesis as described in Guan et al. Nucleic Acid Research, 33:6225-6234 (2005). MBP and MBP fusion proteins were purified as described in the instructions for the pMAL Protein Fusion and Purification System, except in some cases cells were lysed with a lysozyme/detergent solution instead of sonication.
Large-scale purifications were carried out with crude cell extract prepared from 500 to 1000 mL of culture, and loaded on a 2.5 cm diameter column containing 15 ml of amylose resin (NEB #E8021, Ipswich, Mass.). Small-scale purifications were carried out with crude extract prepared from 67 ml of culture, and loaded on a disposable polypropylene column containing 1 ml of amylose resin. SDS-polyacrylamide gel electrophoresis was carried out using 10-20% gradient gels. For quantitation of gel bands, gels were dried between cellophane sheets and scanned using a Microtek III scanner Microtek, Carson, Calif., and densitometry carried out using Image 3 (NIH).
Isolation of Mutants in MBP with Improved Properties
Screening for Improved Yield after Purification:
Random mutagenesis of the malE gene from pMAL-c2x was achieved by error-prone PCR using the primers:
TABLE-US-00001 oligo 1: (SEQ ID NO: 9) 5' GGAGACAUGAATTCAATGAAAATCGAAGAA, and oligo 2: (SEQ ID NO: 10) 5' GGGAAAGUAAGCTTAATCCTTCCCTCGATC
PCR fragments were cloned into linearized pNEB208A using the USER Friendly Cloning Kit, following the manufacturer's instructions. Transformants were grown overnight in 1 mL LB+1 mM IPTG and 100 ug/ml ampicillin, then lysed by adding 0.3 mg/mL lysozyme and 20 units of the S. marscens nuclease, incubating for 10 min, then adding 0.1 ml of 2% Tween 20.
The crude extracts were applied to a 50 uL amylose resin column (NEB #E8021, Ipswich, Mass.) in a Unifilter 800 microplate, and each well was washed with 0.7 ml of 20 mM Tris-Cl, 0.2 M NaCl, 1 mM EDTA, pH 7.4 (column buffer), then with 0.7 mL of 10 mM sodium phosphate, 0.2 M NaCl, 1 mM EDTA, pH 7.2. The protein bound to the amylose resin was then eluted with 0.2 mL of 10 mM maltose, 10 mM sodium phosphate, 0.2 M NaCl, 1 mM EDTA, pH 7.2. The eluate was transferred to an Immulon 2HB microtiter plate (ThermoFisher Scientific, Waltham, Mass.) and incubated overnight at 4° C. The microtiter wells were then emptied, washed twice with 20 mM Tris-CI, 150 mM NaCl, pH 7.5 (TBST), then blocked with 0.36 ml TBST+3% bovine serum albumin for 1 h at 37° C.
The wells were washed twice with TBST, then 0.1 ml of a 1:2000 dilution of anti-MBP monoclonal antibody linked to horse radish peroxidase in TBST+3% bovine serum albumin was added to each well and the plate incubated at 37° C. for 1 h. The wells were emptied, then washed twice with TBST. The wells were developed with 0.01% o-phenylenediamine, 0.003% hydrogen peroxide in water. The detection reaction was stopped by adding 0.025 mL 4 M H2SO4, and wells were assayed spectrophotometrically at 490 nm. Cells were recovered from lysates corresponding to samples that showed higher binding and elution as compared to wild-type MBP. These candidates were grown and retested to confirm the higher binding and elution.
Characterization and Separation of Mutations Obtained after Random Mutagenesis
Two isolates from a library in USER having increased binding and elution profiles were sequenced (FIG. 2). One isolate, G8-1, was found to have a single mis-sense mutation, G1964C, along with a silent mutation. The G1964C mutation corresponds to the amino acid change S146T in MBP. The other isolate, A9 was found to have three mis-sense mutations, A1583G, A2419G and C2465T, along with a silent mutation. The A1583G, A2419G and C2465T mutations correspond to the amino acid changes N195, K298E and A313V, respectively.
Subcloning into pMal-C2X or pSN1578
Each isolate was amplified by PCR with the following primers:
TABLE-US-00002 oligo 3: (SEQ ID NO: 11) 5' GACTCATATGAAAATCGAAGAAGGTAAACTGGTAATCTGGAT TAACGGC and oligo 4: (SEQ ID NO: 12) 5' ATATAAGCTTTCACCTTCCCTCGATCCCGAGGT
The amplified DNA was ethanol precipitated, cut with NdeI and HindIII in NEBuffer 4 (NEB, Ipswich, Mass.), and gel purified. pSN1578 was cleaved with NdeI and HindIII and the vector backbone was gel purified. The G8-1 and A9 fragments were mixed with the pSN1578 fragment and ligated, and the ligation was used to transform TB1. A plasmid preparation from each transformant was sequenced and named pIH1596 for G8-1 and pIH1593 for A9. The 3' primer in this experiment has a stop codon in the correct reading frame to prevent malE translation from proceeding into the lacZα fragment of pMAL. Thus, these subclones produce a modified MBP that ends after the amino acid sequence . . . IEGR encoded by the polylinker. A control plasmid containing a wild-type malE gene followed by a stop codon was constructed by cleaving pMAL-c2X in the polylinker between malE and lacZa with XbaI. The XbaI overhang was filled in using DNA polymerase I, large fragment (Klenow) and all four dNTP's, then the plasmid was recircularized by treatment with T4 ligase. This introduces a stop codon in the same reading frame as malE, and this derivative produces an MBP comparable to that produced by G8-1 and A9, except for an 8 residue extension encoded by the polylinker. This control plasmid was named pKO1483. E. coli TB1 containing pKO1483, pIH1596 and pIH1593 were grown in a 500 mL culture of LB+0.1% glucose and 100 ug/ml ampicillin to 2×108 cells/ml, induced with 0.3 mM IPTG, grown for 2 h at 37° C., then harvested. The cells were resuspended in 25 ml column buffer (0.2 mL of 10 mM maltose, 10 mM sodium phosphate, 0.2 M NaCl, 1 mM EDTA, pH 7.2)+10 mM β-mercaptoethanol, then lysed by sonication. The extract was clarified by centrifuging at 9000×g for 30 min, then diluted 1:4 with column buffer and loaded onto a 15 ml column of amylose resin. The column was washed with about 125 mL column buffer, and eluted with column buffer+10 mM maltose. The yields of MBP were compared among the three strains (FIG. 3). The results confirm that the modified MBPs showed an increased binding to amylose and elution in appropriate buffers.
In order to ascertain which of the three mutation(s) were necessary for increased binding of the A9 variant, the three mutations were subcloned separately into pSN1578, a pMAL-c2G derivative with a deletion internal to the malE gene (which allows easy identification of clones which receive an insert). The A1583G and A2419G mutations either had no effect or reduced the yield of MBP in the affinity purification, and were discarded. The C2465T mutation was recreated in isolation by 4 primer site-directed PCR mutagenesis using pMAL-c2X as the first template, with the primers oligo 5: 5' CTTCAAGGGTCAACCATCCAAACC (SEQ ID NO:13) and oligo 6: 5' AATACGCGGATCTTTCACCAACTCTTC (SEQ ID NO:14) to create the N-terminal PCR fragment, and with primers oligo 7: 5' GAAGAGTTGGTGAAAGATCCGCGTATT (SEQ ID NO:15) and oligo 8: 5' CTGAGAATTCTGAAATCCTTCCCTCGAT (SEQ ID NO:16) to create the C-terminal PCR fragment. The assembly step was carried out with the gel-purified N- and C-terminal fragments as the template and the primers oligo 5 and oligo 8. The final PCR fragment was cut with BlpI and AvaI, gel purified, and ligated to pMAL-c2X that had been cut with BlpI and AvaI and gel purified. The ligation was used to transform TB1, and plasmid was purified from the transformants and sequenced to confirm the C2465T mutation. An isolate was chosen for further study and named pIH1606.
In the construction of pIH1606, the stop codon at the end of MBP was not conserved; this construct expresses MBP fused to the LacZaa fragment. In order to compare the effect of the C2465T mutation to its parent, A9, a stop codon was introduced after the malE gene in pIH1606. The plasmid was cleaved with XbaI, filled in with Klenow plus dNTP's, and religated as described above for pKO1483. The C2465T derivative with a stop codon was called pPR1610. Large scale MBP purifications of TB1 bearing this plasmid, in parallel with pKO1483 and A9, showed that all of the increase in yield of MBP found in A9 could be accounted for by the C2465T mutation. This mutation changes alanine 313 of MBP to a valine (A313V).
In order to be able to compare MBP (S146T) to wild-type MBP and MBP (A313V) in derivatives that have exactly parallel construction, a version of MBP (S146T) was constructed that has the same stop codon as pKO1483 was constructed. An NdeI, BlpI fragment from pIH1596 was purified and subcloned into pKO1493 cut with NdeI and BlpI, creating pIH1619.
Increased Yield of MBP Fusion Proteins
In order to test if the modified MBPs can increase the yield of a fusion protein after purification, the gene encoding the Klenow fragment of E. coli DNA polymerase I was cloned into pMAL-c2X, pIH1619 (S146T) and pPR1610 (A313V). The MBP-Klenow fusion was chosen because it has an inherently low affinity for the amylose column, and during the affinity purification some of the MBP-Klenow protein flows though the amylose column without binding. The Klenow portion of DNA polymerase I was PCR'd from the plasmid pPolA, which contains the BglII-HindIII fragment of the polA gene (Genbank ecopolA:206-4127) cloned into pBR322 between the BglII and HindIII sites. PCR was carried out using the primers oligo 9: 5' CCAGAAGTGACGGCAACGGTGATT ((SEQ ID NO:17) and oligo 10: 5' AAGTGCGGCGACGATAGTCATGCCCCGCGC (SEQ ID NO:18).
The PCR fragment was cleaved with HindIII and ligated to pMAL-c2, which had been cleaved with XmnI and HindIII. The resulting construct was named pIH1040. In order to reduce the chance of PCR errors in the insert, the region from XhoI to HindIII was replaced with the corresponding fragment from pPolA. This construct was checked by sequencing and named pIH1062. The gene encoding the Klenow fragment was cloned into pPR1610 and pIH1619 by PCR using pPolA as a template and the primers oligo 11: 5' GTGATTTCTTATGACAACTACGTCACCATCCTTGATG (SEQ ID NO:19) and oligo 12: 5' TTAAGGATCCTTAGTGCGCCTGATCCCAGT (SEQ ID NO:20)
The PCR fragment was cleaved with BamHI, and the vectors were cleaved with XmnI and BamHI, purified, mixed with the PCR fragment, and ligated. The ligation was used to transform NEB Turbo (Ipswich, Mass.), and transformants were checked for the correct structure by restriction analysis and by expression of the MBP-Klenow fusion and analyzing by SDS-PAGE. The pIH1610-Klenow construct was named pIH1643, and the pIH1619-Klenow construct was named pIH1644.
Affinity purification of MBP-Klenow was performed using cells containing pIH1062, pIH1643 and pIH1644. Crude extract, column flow-through and eluate fractions from each strain were analyzed by SDS-PAGE (FIG. 4). The eluted protein was quantitated by measuring A280 and quantitated using the predicted extinction coefficient of the MBP-Klenow protein (Table 1). Cells bearing pIH1643, carrying the MBP-A313V modification, yielded more than twice as much fusion protein as those containing the wild-type MBP or the S146T modified MBP fusion plasmids.
B. MBP-Chitin Binding Domain:
In order to test if the mutant MBP's ablity to increase the yield of fusion protein from the affinity purification was a general property, another fusion protein that has an inherently low affinity for amylose was tested. The MBP fused to the Bacillus circulans chitin binding domain (MBP-CBD) is encoded by the plasmid pMB50 (FIG. 5). A good fraction of this MBP-CBD fusion protein tends to flow through an amylose column during the affinity purification, similar to MBP-Klenow. The portion of pPR1610 encoding MBP(A313V) was cleaved from the plasmid using HpaI and SacI, and the fragment was purified. pMB50 was cleaved with the same enzymes, the backbone was also purified, and the two fragments were ligated and transformed into ER2523. The resulting plasmid was named pIH1660.
Affinity purification of MBP-CBD was performed using cells containing pMB50 and pIH1660 harvested from 500 mL cultures. The eluted protein was quantitated by measuring A280 and using the predicted extinction coefficient of the MBP-CBD protein (Table 2). Cells bearing pIH1660, carrying the MBP-A313V modification, yielded almost twice as much fusion protein as those containing the wild-type MBP fusion plasmid.
Solubility Enhancement of MBP Fusion Proteins
Construction of MBP Fusions
In order to test whether the modified MBP's retained the ability to enhance the solubility of the protein fused to MBP, two proteins that tend to be insoluble in E. coli, dihydrofolate reductase (DHFR) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were cloned into pIH1619 (S146T) and pIH1610 (A313V). As controls, the same two proteins were cloned into pMAL-c2X and a pMAL-c2G derivative containing the mutations in the Telmer et al. modified MBP named "MBP-DM." The vector encoding MBP-DM was constructed as follows: First, a pMAL-c2G derivative with a translationally silent NsiI site at nucleotide 2030 was constructed by four primer site-directed PCR. The template for both the N- and C-terminal fragments was pMAL-c2X. The primers oligo 13: 5' CCATAGCATATGAAAATCGAAGAAG (SEQ ID NO:21) and oligo 14: 5' CTTGAATGCATAACCCCCGTCAGCAGC (SEQ ID NO:22) were used to create the N-terminal fragment, and the primers oligo 15: 5' GGTTATGCATTCAAGTATGAAAACGGCAAG (SEQ ID NO:23) and oligo 8 were used to create the C-terminal fragment. The PCR fragments were gel purified, then used as a template in the assembly step with the primers oligo 13 and oligo 8. The final PCR fragment was ethanol precipitated, cleaved with NdeI and EcoRI, and gel purified. pMAL-c2X was cleaved with NdeI and EcoRI and gel purified, and the two fragments were mixed and ligated. The ligation was used to transform ER2502, overnight cultures were grown, and miniprep DNA prepared. Several transformants that had acquired the NsiI site and were of the correct size were obtained, but unexpectedly, all of them lacked an EcoRI site. A representative of this set, named pPR1629, was cleaved with NdeI and AvaI, and the malE fragment was gel purified. pSN1578 was cleaved with the same two enzymes and the plasmid backbone was gel purified. These two DNA fragments were ligated, the ligation was used to transform ER2502, and plasmid DNA from one transformant was sequenced, and named pPR1633. Second, the gene for the modified MBP called DM by Telmer and Shilton was constructed by four primer PCR site-directed mutagenesis as follows: The template for both the N- and C-terminal fragments was pMAL-c2X. The primers used for the N-terminal fragment were oligo 16: 5' TATGCATTCAAATACGGTGACATTAAAGACGTGGGCGTGGAT (SEQ ID NO:24) and oligo 17: 5' GCGGCGTTTTCCGCAGTGGCGGCAATACGTGGATCTTTC (SEQ ID NO:25). The C-terminal fragment was produced with the primers oligo 18: 5' GCGGAAAACGCCGCGAAAGGTGAAATCATGCCGAACATC (SEQ ID NO:26) and oligo 8. The assembly PCR was performed using the purified N- and C-terminal fragments with oligo 16 and oligo 8 as the primers. The PCR fragment was ethanol precipitated and ligated to Litmus 38 that had been linearized with EcoRV and treated with Antarctic phosphatase. The ligation was used to transform NEB 5-alpha (Ipswich, Mass.), and plasmid DNA from one transformant was sequenced to confirm the construction and named pPR1638. Plasmid DNAs pPR1633 and pPR1638 were cleaved with NsiI and AvaI, the plasmid backbone from pPR1633 and the `malE fragment from pPR1638 were gel purified, ligated, and the ligation was used to transform NEB Turbo. Plasmid DNA from one transformant was selected for sequencing and named pPR1639.
The DHFR gene was PCR'd using pOTB7-DHFR as a template and using the primers oligo 19: 5' GGATGGTTGGTTCGCTAAACTGCATCGTC (SEQ ID NO:27) and oligo 20: 5' TATTAATCATTCTTCTCATATACTTCAAA (SEQ ID NO:28), then treating the PCR fragment with T4 polymerase and dT for 15 m at room temperature. This treatment produces a short 3' overhang on each end of the fragment, a GG on the upstream end of the DHFR gene, and an A on the downstream end. The vectors pMAL-c2X and pPR1610 were prepared by cleaving with XmnI, then treating with T4 polymerase and dA, which likewise produces a 3' CC overhang at the end of the malE region and a T on the lacZa end. The vector DNAs were ligated to the DHFR fragment and the ligation was used to transform ER1992. For each vector, a transformant was confirmed by restriction analysis and expression of a fusion protein of the expected size analyzed by SDS-PAGE. The pMAL-c2X-DHFR isolate was named pIH1616, and the pPR1610-DHFR isolate was named pIH1617. The DHFR insert was then subcloned into pIH1596 as follows: pIH1616 was cleaved with NdeI and BlpI, the digest run on a gel, and the backbone (including the DHFR gene) was cut out and gel purified. pIH1596 was cut with NdeI and BlpI, and the malE' was gel purified. The vector backbone was ligated to the malE' fragment, and the ligations used to transform ER1992. An isolate was confirmed by restriction analysis and expression of a fusion protein of the expected size analyzed by SDS-PAGE, and named pIH1618. The DHFR insert was subloned into pPR1639 by cleaving both pPR1639 and pIH1616 with AvaI and SalI, gel purifying the vector backbone from pPR1639 and the DHFR fragment from pIH1616, and ligating the two fragments. The ligation was used to transform NEB Turbo, and a transformant was confirmed by sequencing and named pIH1646.
The GAPDH gene was subjected to PCR using pJF931 as a template and using the primers oligo 21: 5' GGATGGTGAAGGTCGGTGTGAACGG (SEQ ID NO:29) and oligo 22: 5' TATTACTCCTTGGAGGCCATGTAGGCCA (SEQ ID NO:30), then treating the PCR fragment with T4 polymerase and dT.
The vectors pMAL-c2X, pPR1610 and pIH1619 were prepared by cleaving with XmnI, then treating with T4 polymerase and dA as described above. The vector DNAs were ligated to the GAPDH fragment and the ligation was used to transform ER1992. For each vector, a transformant was confirmed by restriction analysis and expression of a fusion protein of the expected size analyzed by SDS-PAGE. The pMAL-c2X-GAPDH isolate was named pIH1625, the pPR1610-GAPDH isolate was named pIH1626, and the pIH1619-GAPDH isolate was named pIH1627. The GAPDH insert was then subcloned into pPR1639 by cleaving both pPR1639 and pIH1625 with AvaI and SalI, gel purifying the vector backbone from pPR1639 and the GAPDH fragment from pIH1625, and ligating the two fragments. The ligation was used to transform NEB Turbo, and a transformant was confirmed by restriction analysis and expression of a fusion protein of the expected size analyzed by SDS-PAGE, and named pIH1645.
A 20 ml culture of TB1 containing the eight pMAL plasmids ((pMAL-c2X, pIH1619 (carrying S146T) and pPR1610 (carrying A313V), each plasmid further containing a DNA encoding DHFR or GApDH)) was grown to 2×108/ml in LB amp, induced with 0.3 mM IPTG, incubated for an additional two hours, then cells were harvested by centrifugation at 3000×g in a microfuge. Each pellet was resuspended in 2 ml of 50 mM Tris-CI, pH 7.9, 50 mM NaCl, 0.75 mM EDTA, 0.6% Mega 10, 150 ug/ml lysozyme, and 20 Kunitz units/ml Serracia marscesens nuclease, and incubated at room temperature for 10 m. The resuspended pellet was designated the total cell extract. A sample of 125 ul was removed and centrifuged for 2 m at 14,000×g. The supernatant was removed and designated the soluble fraction. The pellet was resupended in 125 ul of the same buffer, and designated the insoluble fraction. A sample (5 ul) of each fraction was run on SDS-PAGE for each strain (FIG. 5). The gels were dried and scanned, and the amount of MBP fusion protein in each lane was quantitated as a ratio of soluble protein to protein present in the cell lysate before centrifugation (Table 2). For both DHFR and GAPDH fusions, the A313V and S146T modified MBPs enhanced the solubility of the fusion protein as compared to wild-type MBP. Fusions made with the DM modified MBP, as expected, showed reduced solubility compared to wild-type MBP.
Use of an MBP Mutant in K. lactis
Construction of a K. lactis MBP(A313V)-fusion Expression Vector
The gene encoding the mutant maltose-binding protein, MBP(A313V), was amplified by PCR using the forward and reverse primers, respectively: oligo 23: 5' GCCCAAGCTTGCCACCATGAAAATCGAAGAAGGT (SEQ ID NO:31) and oligo 24: 5' GCGCTCGAGCTTGTCATCGTCATCCGAGCTCGAATTAGTCTGCGC (SEQ ID NO:32). The forward primer was engineered to contain a HindIII restriction enzyme site (bold text) followed by the Kozak consensus sequence (italics) that immediately preceeds the malE gene initiation codon (underlined). The reverse primer was engineered to contain a XhoI restriction enzyme site (bold text) immediately followed by DNA encoding the proteolytic recognition site of the protease enterokinase (italic and underlined text). No stop codon was incorporated onto the reverse primer to allow for the construction of in-frame C-terminal MBP(A313V) fusion expression cassettes. The malE gene was amplified from the plasmid pPR1610 containing the full-length gene, using Phusion polymerase. The amplified gene was cloned into the HindIII and XhoI restriction sites of the K. lactis expression vector pKLAC1 to create a K. lactis MBP(A313V)-fusion expression vector (pKIMF2). This cloning strategy results in the replacement of the K. lactis α-mating factor pre-pro signal sequence in pKLAC1 with the malE gene. Thus the MBP- and MBP(A313V)-fusion proteins will not be directed to the secretory pathway but instead will be retained in the yeast cytosol.
Expression and Purification of an MBP(A313V)-Fusion Protein in K. lactis
The gene encoding a truncated form of paramyosin (Steel et al., J. Immunol. 145:3917-3923 (1990)) was amplified by PCR using the following primers: oligo 25: 5'GCGCTCGAGAATTCCGCATTCGGTAGTATG (SEQ ID NO:33) and oligo 26: 5'ATAAGAATGCGGCCGCTCACGACGTTGTAAAACGACGGCCAGT (SEQ ID NO:34). The forward primer was engineered to contain a XhoI restriction site (bold text). The reverse primer was engineered to contain a NotI restriction site (bold text) immediately upstream of the PMΔSal stop codon (italic text).
The cloning strategy was as follows. The ˜750 bp paramyosin gene was amplified and purified. The fragment was double-digested with XhoI and NotI and cloned into the XhoI and NotI restriction sites of the expression vector, pKIMF2 creating an in-frame fusion between the C-terminus and the N-terminus of PMΔSal. The plasmid constructed in this way was named pKLMF2-PMΔSal (FIG. 7).
The MBP(A313V)-PMΔSal fusion vector (pKIMF2-PMΔSal) was linearized by SacII restriction digestion and the purified product was transformed into chemically competent K. lactis cells using the K. lactis Protein Expression Kit according its instructions. Transformant colonies were selected on acetamide plates and clones containing the correctly integrated MBP(A313V)-PMΔSaI expression cassette were identified by whole cell PCR as described in the above kit.
A 20 ml YPGalactose medium (1% yeast extract, 2% peptone, 2% galactose) culture was inoculated with a strain of K. lactis cells containing a multi-copy integrated MBP(A313V)-PMΔSal expression cassette and grown at 30° C. in an incubator with a shaking platform for 4 days. The total cell content of the culture after growth was determined to be approximately 8.2×1010 cells (or approximately 4.1×108 cells/ml). Cells were harvested by centrifugation at 8,000 rpm for 10 minutes at 4° C. The cells were washed once in distilled water to remove the excess media.
For the preparation of a lysate, the cells were re-suspended in a 2 ml solution of 10 mg/ml lyticase in 1M sorbitol and incubated at 37° C. for 1 hour. Cells were harvested by gentle centrifugation (7,000 r.p.m.) in a micro-centrifuge for 2 minutes. Cell pellets were resuspended in a total volume of 3 ml ice-cold amylose column buffer (20 mM Tris-CI ph 7.4, 0.2 M NaCl, 1 mM EDTA) containing a protease inhibitor cocktail (Complete® protease inhibitor cocktail, Roche). The cell slurry was transferred to a glass tube and placed on ice. An equal volume of acid washed glass beads (425-600 micron, Sigma) were added to the cell slurry and cells were broken by vortexing 10 times, each for a 1 minute duration. Cell lysate was transferred to a new tube and the glass beads washed 4 times with 1 ml column buffer. The cell lysate and washes were pooled and the cellular debris was removed by centrifugation. Cleared cell lysate was diluted to 8 ml with column buffer.
For fusion protein purification, the cell lysate was passed over a 1.6 ml amylose resin column pre-equilibrated with 15 ml of column buffer. The column was washed with 24 ml of column buffer and bound protein was eluted in 0.4 ml fractions with column buffer containing 10 mM maltose. Cell lysate and purified proteins were resolved by SDS-PAGE on a 10-20% Tris-Glycine gradient gel. Proteins were identified by SimplyBlue Safestain. FIG. 6 shows that the major eluted protein corresponds to the size expected (68.5 kDa) for an MBP(A313V)-PMΔSal fusion protein.
TABLE-US-00003 TABLE 1 Yield of MBP-Klenow for wild-type and modified MBP's plasmid MBP Yield pIH1062 WT 0.7 mgs pIH1643 A313V 2 mgs pIH1644 S146T 0.7 mgs
TABLE-US-00004 TABLE 2 Yield of MBP-CBD for wild-type and MBP(A313V) plasmid MBP Yield pMB50 WT 13.2 mgs pIH1660 A313V 21.8 mgs
TABLE-US-00005 TABLE 3 Solubility of MBP fusion proteins Plasmid Fusion protein % Soluble pIH1616 MBP-DHFR 36% pIH1617 MBP(A313V)-DHFR 67% pIH1618 MBP(S146T)-DHFR 67% pIH1646 MBP(DM)-DHFR 25% pIH1625 MBP-GAPDH 42% pIH1626 MBP(A313V)-GAPDH 59% pIH1627 MBP(S146T)-GAPDH 53% pIH1645 MBP(DM)-GAPDH 17%
3411188DNAunknownDNA encoding wild-type maltose binding protein 1gcacttcacc aacaaggacc atagcatatg aaaatcgaag aaggtaaact ggtaatctgg 60attaacggcg ataaaggcta taacggtctc gctgaagtcg gtaagaaatt cgagaaagat 120accggaatta aagtcaccgt tgagcatccg gataaactgg aagagaaatt cccacaggtt 180gcggcaactg gcgatggccc tgacattatc ttctgggcac acgaccgctt tggtggctac 240gctcaatctg gcctgttggc tgaaatcacc ccggacaaag cgttccagga caagctgtat 300ccgtttacct gggatgccgt acgttacaac ggcaagctga ttgcttaccc gatcgctgtt 360gaagcgttat cgctgattta taacaaagat ctgctgccga acccgccaaa aacctgggaa 420gagatcccgg cgctggataa agaactgaaa gcgaaaggta agagcgcgct gatgttcaac 480ctgcaagaac cgtacttcac ctggccgctg attgctgctg acgggggtta tgcgttcaag 540tatgaaaacg gcaagtacga cattaaagac gtgggcgtgg ataacgctgg cgcgaaagcg 600ggtctgacct tcctggttga cctgattaaa aacaaacaca tgaatgcaga caccgattac 660tccatcgcag aagctgcctt taataaaggc gaaacagcga tgaccatcaa cggcccgtgg 720gcatggtcca acatcgacac cagcaaagtg aattatggtg taacggtact gccgaccttc 780aagggtcaac catccaaacc gttcgttggc gtgctgagcg caggtattaa cgccgccagt 840ccgaacaaag agctggcaaa agagttcctc gaaaactatc tgctgactga tgaaggtctg 900gaagcggtta ataaagacaa accgctgggt gccgtagcgc tgaagtctta cgaggaagag 960ttggcgaaag atccacgtat tgccgccact atggaaaacg cccagaaagg tgaaatcatg 1020ccgaacatcc cgcagatgtc cgctttctgg tatgccgtgc gtactgcggt gatcaacgcc 1080gccagcggtc gtcagactgt cgatgaagcc ctgaaagacg cgcagactaa ttcgagctcg 1140aacaacaaca acaataacaa taacaacaac ctcgggatcg agggaagg 11882136PRTunknownwild-type maltose binding protein 2Leu Glu Arg Trp Glu Ile Ser Glu Glu Lys Leu Thr Asp Leu Val Asp1 5 10 15Lys Asn Pro Ser Leu Arg Gly Met Ile Leu Gly Tyr Val Ala Glu Asp 20 25 30Lys Phe His Glu Leu Phe Leu Glu Asp Glu Arg Val Lys Glu Val Ser 35 40 45Lys Asp Asp Asp His Asp Arg Lys Lys Lys Gly Asp Arg Thr Phe Ile 50 55 60Tyr Lys Gly Lys Lys Phe Thr Val Glu Val Lys Ser Leu Gln Thr Ala65 70 75 80Met Cys Lys Lys Asn Glu Asp Gly Thr Tyr Ser Gly Lys Ala Gln Val 85 90 95Asp Gly Ser Asp Arg Arg Ile Val Lys Phe Pro Asp Asn Ser Glu Leu 100 105 110Asn Thr Thr Leu Leu Leu Lys Gly Glu Phe Asp Leu Leu Ala Val Asn 115 120 125Cys Phe Ala Phe Gly Glu Gly Trp 130 13531188DNAunknownDNA sequence encoding the maltose binding protein mutant A9 3gcacttcacc aacaaggacc atagcatatg aaaatcgaag aaggtaaact ggtaatctgg 60attaacggcg ataaaggcta tagcggtctc gctgaagtcg gtaagaaatt cgagaaagat 120accggaatta aagtcaccgt tgagcatccg gataaactgg aagagaaatt cccacaggtt 180gcggcaactg gcgatggccc tgacattatc ttctgggcac acgaccgctt tggtggctac 240gctcaatctg gcctgttggc tgaaatcacc ccggacaaag cgttccagga caagctgtat 300ccgtttacct gggatgccgt acgttacaac ggcaagctga ttgcttaccc gatcgctgtt 360gaagcgttat cgctgattta taacaaagat ctgctgccga acccgccaaa aacctgggaa 420gagatcccgg cgctggataa agaactgaaa gcgaaaggta agagcgcgct gatgttcaac 480ctgcaagaac cgtacttcac ctggccgctg attgctgctg acgggggtta tgcgttcaag 540tatgaaaacg gcaagtacga cattaaagac gtgggcgtgg ataacgctgg cgcgaaagcg 600ggtctgacct tcctggttga cctgattaag aacaaacaca tgaatgcaga caccgattac 660tccatcgcag aagctgcctt taataaaggc gaaacagcga tgaccatcaa cggcccgtgg 720gcatggtcca acatcgacac cagcaaagtg aattatggtg taacggtact gccgaccttc 780aagggtcaac catccaaacc gttcgttggc gtgctgagcg caggtattaa cgccgccagt 840ccgaacaaag agctggcaaa agagttcctc gaaaactatc tgctgactga tgaaggtctg 900gaagcggtta ataaagacga accgctgggt gccgtagcgc tgaagtctta cgaggaagag 960ttggtgaaag atccacgtat tgccgccact atggaaaacg cccagaaagg tgaaatcatg 1020ccgaacatcc cgcagatgtc cgctttctgg tatgccgtgc gtactgcggt gatcaacgcc 1080gccagcggtc gtcagactgt cgatgaagcc ctgaaagacg cgcagactaa ttcgagctcg 1140aacaacaaca acaataacaa taacaacaac ctcgggatcg agggaagg 11884387PRTunknownmaltose binding protein mutant A9 4Met Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys1 5 10 15Gly Tyr Ser Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr 20 25 30Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe 35 40 45Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala 50 55 60His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile65 70 75 80Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp 85 90 95Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu 100 105 110Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys 115 120 125Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly 130 135 140Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro145 150 155 160Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys 165 170 175Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly 180 185 190Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp 195 200 205Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala 210 215 220Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys225 230 235 240Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser 245 250 255Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro 260 265 270Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp 275 280 285Glu Gly Leu Glu Ala Val Asn Lys Asp Glu Pro Leu Gly Ala Val Ala 290 295 300Leu Lys Ser Tyr Glu Glu Glu Leu Val Lys Asp Pro Arg Ile Ala Ala305 310 315 320Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln 325 330 335Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala 340 345 350Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Asn 355 360 365Ser Ser Ser Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Leu Gly Ile 370 375 380Glu Gly Arg38551188DNAunknownDNA sequence encoding the maltose binding protein mutant G8-1 5gcacttcacc aacaaggacc atagcatatg aaaatcgaag aaggtaaact ggtaatctgg 60attaacggcg ataaaggcta taacggtctc gctgaagtcg gtaagaaatt cgagaaagat 120accggaatta aagtcaccgt tgagcatccg gataaactgg aagagaaatt cccacaggtt 180gcggcaactg gcgatggccc tgacattatc ttctgggcac acgaccgctt tggtggctac 240gctcaatctg gcctgttggc tgaaatcacc ccggacaaag cgttccagga caagctgtat 300ccgtttacct gggatgccgt acgttacaac ggcaagctga ttgcttaccc gatcgctgtt 360gaagcgttat cgctgattta taacaaagat ctgctgccga acccgccaaa aacctgggaa 420gagatcccgg cgctggataa agaactgaaa gcgaaaggta agaccgcgct gatgttcaac 480ctgcaagaac cgtacttcac ctggccgctg attgctgctg acgggggcta tgcgttcaag 540tatgaaaacg gcaagtacga cattaaagac gtgggcgtgg ataacgctgg cgcgaaagcg 600ggtctgacct tcctggttga cctgattaaa aacaaacaca tgaatgcaga caccgattac 660tccatcgcag aagctgcctt taataaaggc gaaacagcga tgaccatcaa cggcccgtgg 720gcatggtcca acatcgacac cagcaaagtg aattatggtg taacggtact gccgaccttc 780aagggtcaac catccaaacc gttcgttggc gtgctgagcg caggtattaa cgccgccagt 840ccgaacaaag agctggcaaa agagttcctc gaaaactatc tgctgactga tgaaggtctg 900gaagcggtta ataaagacaa accgctgggt gccgtagcgc tgaagtctta cgaggaagag 960ttggcgaaag atccacgtat tgccgccact atggaaaacg cccagaaagg tgaaatcatg 1020ccgaacatcc cgcagatgtc cgctttctgg tatgccgtgc gtactgcggt gatcaacgcc 1080gccagcggtc gtcagactgt cgatgaagcc ctgaaagacg cgcagactaa ttcgagctcg 1140aacaacaaca acaataacaa taacaacaac ctcgggatcg agggaagg 11886387PRTunknownmaltose binding protein mutant G8-1 6Met Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys1 5 10 15Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr 20 25 30Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe 35 40 45Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala 50 55 60His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile65 70 75 80Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp 85 90 95Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu 100 105 110Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys 115 120 125Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly 130 135 140Lys Thr Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro145 150 155 160Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys 165 170 175Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly 180 185 190Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp 195 200 205Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala 210 215 220Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys225 230 235 240Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser 245 250 255Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro 260 265 270Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp 275 280 285Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala 290 295 300Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala Ala305 310 315 320Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln 325 330 335Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala 340 345 350Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Asn 355 360 365Ser Ser Ser Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Leu Gly Ile 370 375 380Glu Gly Arg38576835DNAunknownpMB50 7ccgacaccat cgaatggtgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga 60gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg 120gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa 180cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac 240aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc 300acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg 360tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc 420ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca 480ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga 540cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc 600tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg 660cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag 720cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga 780atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa 840tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg 900acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc 960gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga 1020agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata 1080cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt 1140cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag 1200gcacaattct catgtttgac agcttatcat cgactgcacg gtgcaccaat gcttctggcg 1260tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc gtaaatcact gcataattcg 1320tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt 1380ctggcaaata ttctgaaatg agctgttgac aattaatcat cggctcgtat aatgtgtgga 1440attgtgagcg gataacaatt tcacacagga aacagccagt ccgtttaggt gttttcacga 1500gcacttcacc aacaaggacc atagattatg aaaatcgaag aaggtaaact ggtaatctgg 1560attaacggcg ataaaggcta taacggtctc gctgaagtcg gtaagaaatt cgagaaagat 1620accggaatta aagtcaccgt tgagcatccg gataaactgg aagagaaatt cccacaggtt 1680gcggcaactg gcgatggccc tgacattatc ttctgggcac acgaccgctt tggtggctac 1740gctcaatctg gcctgttggc tgaaatcacc ccggacaaag cgttccagga caagctgtat 1800ccgtttacct gggatgccgt acgttacaac ggcaagctga ttgcttaccc gatcgctgtt 1860gaagcgttat cgctgattta taacaaagat ctgctgccga acccgccaaa aacctgggaa 1920gagatcccgg cgctggataa agaactgaaa gcgaaaggta agagcgcgct gatgttcaac 1980ctgcaagaac cgtacttcac ctggccgctg attgctgctg acgggggtta tgcgttcaag 2040tatgaaaacg gcaagtacga cattaaagac gtgggcgtgg ataacgctgg cgcgaaagcg 2100ggtctgacct tcctggttga cctgattaaa aacaaacaca tgaatgcaga caccgattac 2160tccatcgcag aagctgcctt taataaaggc gaaacagcga tgaccatcaa cggcccgtgg 2220gcatggtcca acatcgacac cagcaaagtg aattatggtg taacggtact gccgaccttc 2280aagggtcaac catccaaacc gttcgttggc gtgctgagcg caggtattaa cgccgccagt 2340ccgaacaaag agctggcaaa agagttcctc gaaaactatc tgctgactga tgaaggtctg 2400gaagcggtta ataaagacaa accgctgggt gccgtagcgc tgaagtctta cgaggaagag 2460ttggcgaaag atccacgtat tgccgccacc atggaaaacg cccagaaagg tgaaatcatg 2520ccgaacatcc cgcagatgtc cgctttctgg tatgccgtgc gtactgcggt gatcaacgcc 2580gccagcggtc gtcagactgt cgatgaagcc ctgaaagacg cgcagactaa ttcgagctcg 2640aacaacaaca acaataacaa taacaacaac ctcgggatcg agggaagggg tacgctcgag 2700ggttctcagc atgcaccggg tggcctgacc ggtctgaact caggcctcac gacaaatcct 2760ggtgtatccg cttggcaggt caacacagct tatactgcgg gacaattggt cacatataac 2820ggcaagacgt ataaatgttt gcagccccac acctccttgg caggatggga accatccaac 2880gttcctgcct tgtggcagct tcaatgactg caggcaagct tggcactggc cgtcgtttta 2940caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc 3000cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg 3060cgcagcctga atggcgaatg gcagcttggc tgttttggcg gatgagataa gattttcagc 3120ctgatacaga ttaaatcaga acgcagaagc ggtctgataa aacagaattt gcctggcggc 3180agtagcgcgg tggtcccacc tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc 3240gatggtagtg tggggtctcc ccatgcgaga gtagggaact gccaggcatc aaataaaacg 3300aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct 3360cctgagtagg acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg 3420gtggcgggca ggacgcccgc cataaactgc caggcatcaa attaagcaga aggccatcct 3480gacggatggc ctttttgcgt ttctacaaac tctttttgtt tatttttcta aatacattca 3540aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg 3600aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc 3660cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg 3720ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt 3780cgccccgaag aacgttctcc aatgatgagc acttttaaag ttctgctatg tggcgcggta 3840ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat 3900gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga 3960gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca 4020acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact 4080cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc 4140acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact 4200ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt 4260ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt 4320gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt 4380atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata 4440ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag 4500attgatttac cccggttgat aatcagaaaa gccccaaaaa caggaagatt gtataagcaa 4560atatttaaat tgtaaacgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca 4620gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaatagc 4680ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg 4740actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat 4800cacccaaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag 4860ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga 4920agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa 4980ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtaaaaggat ctaggtgaag 5040atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg 5100tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc 5160tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag 5220ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc 5280cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac 5340ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc 5400gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt 5460tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt 5520gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc 5580ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
ggggaaacgc ctggtatctt 5640tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca 5700ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt 5760tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt 5820attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag 5880tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc 5940ggtatttcac accgcatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta 6000agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc 6060caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag 6120ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg 6180cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgcag cgattcacag atgtctgcct 6240gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa 6300agcgggccat gttaagggcg gttttttcct gtttggtcac ttgatgcctc cgtgtaaggg 6360ggaatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg 6420ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt 6480atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac 6540agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat 6600ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat 6660tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg 6720tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa 6780cgacaggagc acgatcatgc gcacccgtgg ccaggaccca acgctgcccg aaatt 6835810632DNAunknownpKLIMF2-PMdeltaSal 8aagcttgcca ccatgaaaat cgaagaaggt aaactggtaa tctggattaa cggcgataaa 60ggctataacg gtctcgctga agtcggtaag aaattcgaga aagataccgg aattaaagtc 120accgttgagc atccggataa actggaagag aaattcccac aggttgcggc aactggcgat 180ggccctgaca ttatcttctg ggcacacgac cgctttggtg gctacgctca atctggcctg 240ttggctgaaa tcaccccgga caaagcgttc caggacaagc tgtatccgtt tacctgggat 300gccgtacgtt acaacggcaa gctgattgct tacccgatcg ctgttgaagc gttatcgctg 360atttataaca aagatctgct gccgaacccg ccaaaaacct gggaagagat cccggcgctg 420gataaagaac tgaaagcgaa aggtaagagc gcgctgatgt tcaacctgca agaaccgtac 480ttcacctggc cgctgattgc tgctgacggg ggttatgcgt tcaagtatga aaacggcaag 540tacgacatta aagacgtggg cgtggataac gctggcgcga aagcgggtct gaccttcctg 600gttgacctga ttaaaaacaa acacatgaat gcagacaccg attactccat cgcagaagct 660gcctttaata aaggcgaaac agcgatgacc atcaacggcc cgtgggcatg gtccaacatc 720gacaccagca aagtgaatta tggtgtaacg gtactgccga ccttcaaggg tcaaccatcc 780aaaccgttcg ttggcgtgct gagcgcaggt attaacgccg ccagtccgaa caaagagctg 840gcaaaagagt tcctcgaaaa ctatctgctg actgatgaag gtctggaagc ggttaataaa 900gacaaaccgc tgggtgccgt agcgctgaag tcttacgagg aagagttggt gaaagatcca 960cgtattgccg ccactatgga aaacgcccag aaaggtgaaa tcatgccgaa catcccgcag 1020atgtccgctt tctggtatgc cgtgcgtact gcggtgatca acgccgccag cggtcgtcag 1080actgtcgatg aagccctgaa agacgcgcag actaattcga gctcggatga cgatgacaag 1140ctcgagaatt ccgcattcgg tagtatgtcg gttgccgatt tgggaagttt aactcgcttg 1200gaggacaaga ttcgtctact gcaagaagat ctcgaatccg aacgtgaact tcgaaataga 1260atcgaacgag aaagagctga tcttagtgta caactgattg cattaactga tagacttgaa 1320gatgctgagg gtactactga tagtcagatt gaatcaaatc gtaaacgtga agcagaattg 1380caaaaattac gtaaattatt ggaagaatca caattagaaa atgaagatgc aatgaatgtt 1440ttacgtaaaa agcatcaaga tgcatgtctc gattacgctg aacaaattga acaattacaa 1500aagaaaaatt caaagattga tcgtgaacgt caacgtctgc aacatgaagt aattgagctt 1560actgcgacaa ttgatcaact tcaaaaggat aagcatttgg cggaaaaagc agcggaacgt 1620tttgaagcgc aaactatcga attgagtaat aaagttgaag atttaaatcg acatgttaat 1680gatttagctc aacaacgtca acgtttacaa gctgaaaata acgatcttct caaagagatt 1740catgatcaaa aagtacaatt ggataatttg caacacgtga aatatcaact tgcgcaacaa 1800cttgaagaag cacgtcgacc tgcaggcaag cttggcactg gccgtcgttt tacaacgtcg 1860tgagcggccg cttaattaag gccttgaatc gagaatttat acttagataa gtatgtactt 1920acaggtatat ttctatgaga tactgatgta tacatgcatg ataatattta aacggttatt 1980agtgccgatt gtcttgtgcg ataatgacgt tcctatcaaa gcaatacact taccacctat 2040tacatgggcc aagaaaatat tttcgaactt gtttagaata ttagcacaga gtatatgatg 2100atatccgtta gattatgcat gattcattcc tacaactttt tcgtagcata aggattaatt 2160acttggatgc caataaaaaa aaaaaacatc gagaaaattt cagcatgctc agaaacaatt 2220gcagtgtatc aaagtaaaaa aaagattttc actacatgtt ccttttgaag aaagaaaatc 2280atggaacatt agatttacaa aaatttaacc accgctgatt aacgattaga ccgttaagcg 2340cacaacaggt tattagtaca gagaaagcat tctgtggtgt tgccccggac tttcttttgc 2400gacataggta aatcgaatac catcatacta tcttttccaa tgactcccta aagaaagact 2460cttcttcgat gttgtatacg ttggagcata gggcaagaat tgtggcttga gatcatcctt 2520ttgttgtttc cgggtgtaca atatggactt cctcttttct ggcaaccaaa cccatacatc 2580gggattccta taataccttc gttggtctcc ctaacatgta ggtggcggag gggagatata 2640caatagaaca gataccagac aagacataat gggctaaaca agactacacc aattacactg 2700cctcattgat ggtggtacat aacgaactaa tactgtagcc ctagacttga tagccatcat 2760catatcgaag tttcactacc ctttttccat ttgccatcta ttgaagtaat aataggcgca 2820tgcaacttct tttctttttt tttcttttct ctctcccccg ttgttgtctc accatatccg 2880caatgacaaa aaaatgatgg aagacactaa aggaaaaaat taacgacaaa gacagcacca 2940acagatgtcg ttgttccaga gctgatgagg ggtatctcga agcacacgaa actttttcct 3000tccttcattc acgcacacta ctctctaatg agcaacggta tacggccttc cttccagtta 3060cttgaatttg aaataaaaaa aagtttgctg tcttgctatc aagtataaat agacctgcaa 3120ttattaatct tttgtttcct cgtcattgtt ctcgttccct ttcttccttg tttctttttc 3180tgcacaatat ttcaagctat accaagcata caatcaagga attccggatc cgccaccatg 3240cctcaatcct gggaagaact ggccgctgat aagcgcgccc gcctcgcaaa aaccatccct 3300gatgaatgga aagtccagac gctgcctgcg gaagacagcg ttattgattt cccaaagaaa 3360tcggggatcc tttcagaggc cgaactgaag atcacagagg cttccgctgc ggatcttgtg 3420tccaagctgg cggccggaga gttgacctcg gtggaagtta cgctagcatt ctgtaaacgg 3480gcagcaatcg cccagcagtt aacaaactgc gcccacgagt tcttccctga cgccgctctc 3540gcgcaggcaa gggaactcga tgaatactac gcaaagcaca agagacccgt tggtccactc 3600catggcctcc ccatctctct caaagaccag cttcgagtca agggctacga aacatcaatg 3660ggctacatct catggctaaa caagtacgac gaaggggact cggttctgac aaccatgctc 3720cgcaaagccg gtgccgtctt ctacgtcaag acctctgtcc cgcagaccct gatggtctgc 3780gagacagtca acaacatcat cgggcgcacc gtcaacccac gcaacaagaa ctggtcgtgc 3840ggcggcagtt ctggtggtga gggtgcgatc gttgggattc gtggtggcgt catcggtgta 3900ggaacggata tcggtggctc gattcgagtg ccggccgcgt tcaacttcct gtacggtcta 3960aggccgagtc atgggcggct gccgtatgca aagatggcga acagcatgga gggtcaggag 4020acggtgcaca gcgttgtcgg gccgattacg cactctgttg aggacctccg cctcttcacc 4080aaatccgtcc tcggtcagga gccatggaaa tacgactcca aggtcatccc catgccctgg 4140cgccagtccg agtcggacat tattgcctcc aagatcaaga acggcgggct caatatcggc 4200tactacaact tcgacggcaa tgtccttcca caccctccta tcctgcgcgg cgtggaaact 4260accgtcgccg cactcgccaa agccggtcac accgtgaccc cgtggacgcc atacaagcac 4320gatttcggcc acgatctcat ctcccatatc tacgcggctg acggcagcgc cgacgtaatg 4380cgcgatatca gtgcatccgg cgagccggcg attccaaata tcaaagacct actgaacccg 4440aacatcaaag ctgttaacat gaacgagctc tgggacacgc atctccagaa gtggaattac 4500cagatggagt accttgagaa atggcgggag gctgaagaaa aggccgggaa ggaactggac 4560gccatcatcg cgccgattac gcctaccgct gcggtacggc atgaccagtt ccggtactat 4620gggtatgcct ctgtgatcaa cctgctggat ttcacgagcg tggttgttcc ggttaccttt 4680gcggataaga acatcgataa gaagaatgag agtttcaagg cggttagtga gcttgatgcc 4740ctcgtgcagg aagagtatga tccggaggcg taccatgggg caccggttgc agtgcaggtt 4800atcggacgga gactcagtga agagaggacg ttggcgattg cagaggaagt ggggaagttg 4860ctgggaaatg tggtgactcc atagcccggg gggggctcga tcccctcgcg agttggttca 4920gctgctgcct gaggctggac gacctcgcgg agttctaccg gcagtgcaaa tccgtcggca 4980tccaggaaac cagcagcggc tatccgcgca tccatgcccc cgaactgcag gagtggggag 5040gcacgatggc cgctttggtc gatctagatt acgtggaaga aaggtagtaa aagtagtagt 5100ataagtagta aaaagaggta aaaagagaaa accggctaca tactagagaa gcacgtacac 5160aaaaactcat aggcacttca tcatacgaca gtttcttgat gcattataat agtgtattag 5220atattttcag aaatatgcat agaacctcct cttgccttta ctttttatac atagaacatt 5280ggcagattta cttacactac tttgtttcta cgccatttct tttgttttca acacttagac 5340aagttgttga gaaccggact actaaaaagc aatgttccca ctgaaaatca tgtacctgca 5400ggataataac cccctaattc tgcatcgatc cagtatgttt ttttttctct actcattttt 5460acctgaagat agagcttcta aaacaaaaaa aatcagtgat tacatgcata ttgtgtgttc 5520tagtaaccaa aggaaaggaa cagatagata aaattccgag actgtcaaat taggtttttt 5580tctttttttt tggcgggagt cagtgggccg aaatatgttc ttggcctaga acttaatctg 5640gtttgatcat gccaatactt gcctgagtgc ccgacttttt gcccaccctc ttgccttctg 5700tcatccttca aaacccacct gttttccagc cgtatcttcg ctcgcatcta cacatactgt 5760gccatatctt gtgtgtagcc ggacgtgact atgaccaaaa acaaacaagg agaactgttc 5820gccgatttgt aacactcctg catccatcca agtgggtatg cgctatgcaa tgttaagcta 5880ggtcaggtca gaccaggtcc aaggacagca acttgactgt atgcaacctt taccatcttt 5940gcacagaaca tacttgtagc tagctagtta cacttatgga ccgaaaaggc accccaccat 6000gtctgtccgg ctttagagta cggccgcaga ccgctgattt gccttgccaa gcagtagtca 6060caatgcatcg catgagcaca cgggcacggg cacgggcaca ggaaccattg gcaaaaatac 6120cagatacact ataccgacgt atatcaagcc caagtttaaa attcctaaat ttccgcggct 6180acttttcaat tccctatagt gagtcgtatt aaattcgtaa tcatgtcata gctgtttcct 6240gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt 6300aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc 6360gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg 6420agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 6480gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 6540gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 6600cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 6660aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 6720tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 6780ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat 6840ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 6900cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 6960ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 7020gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt 7080atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 7140aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga 7200aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 7260gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc 7320cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct 7380gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca 7440tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct 7500ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca 7560ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc 7620atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg 7680cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct 7740tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa 7800aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta 7860tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc 7920ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg 7980agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa 8040gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg 8100agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc 8160accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg 8220gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat 8280cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata 8340ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg cgccctgtag cggcgcatta 8400agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg 8460cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa 8520gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc 8580aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt 8640cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca 8700acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc 8760tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta 8820acgcttacaa tttccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc 8880gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt 8940gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt gccaagctcc 9000cgcggggatc gactcataaa atagtaacct tctaatgcgt atctattgac taccaaccat 9060tagtgtggtt gcagaaggcg gaattctccc ttcttcgaat tcagcttgct ttttcatttt 9120ttattttcca tttttcagtt tttgtttgtg tcgaatttag ccagttgctt ctccaagatg 9180aaaaaaaccc ctgcgcagtt tctgtgctgc aagatcctaa tcgacttttc caccccccac 9240aaaagtaaat gttcttttgt tacattcgcg tgggtagcta gctccccgaa tcttcaaagg 9300acttagggac tgcactacat cagagtgtgt tcacctggtt tgctgcctgg tttgaaagaa 9360aagagcaggg aactcgcggg ttcccggcga ataatcatgc gatagtcctt tggccttcca 9420agtcgcatgt agagtagaca acagacaggg agggcaggaa ggatctttca ctgagatcct 9480gtatcttgtt gggtaagtcg gatgaaaggg gaatcgtatg agattggaga ggatgcggaa 9540gaggtaacgc cttttgttaa cttgtttaat tattatgggg caggcgagag ggggaggaat 9600gtatgtgtgt gaggcgggcg agacggagcc atccaggcca ggtagaaata gagaaagccg 9660aatgttagac aatatggcag cgtagtagag taggtaggta ggcaagtact gctagcaaag 9720aggagaaggg taagctcact cttcgcattc cacaccgtta gtgtgtcagt ttgaacaaaa 9780aaacaatcat cataccaatt gatggactgt ggactggctt ttggaacggc ttttcggact 9840gcgattattc gtgaggaatc aaggtaggaa tttggtcata tttacggaca acagtgggtg 9900attcccatat ggagtaggaa aacgagatca tggtatcctc agatatgttg cggaattctg 9960ttcaccgcaa agttcagggt gctctggtgg gtttcggttg gtctttgctt tgcttctccc 10020ttgtcttgca tgttaataat agcctagcct gtgagccgaa acttagggta ggcttagtgt 10080tggaacgtac atatgtatca cgttgacttg gtttaaccag gcgacctggt agccagccat 10140acccacacac gttttttgta tcttcagtat agttgtgaaa agtgtagcgg aaatttgtgg 10200tccgagcaac agcgtctttt tctagtagtg cggtcggtta cttggttgac attggtattt 10260ggactttgtt gctacaccat tcactacttg aagtcgagtg tgaagggtat gatttctagt 10320ggtgaacacc tttagttacg taatgttttc attgctgttt tacttgagat ttcgattgag 10380aaaaaggtat ttaatagctc gaatcaatgt gagaacagag agaagatgtt cttccctaac 10440tcgaaaggta tatgaggctt gtgtttctta ggagaattat tattcttttg ttatgttgcg 10500cttgtagttg gaaaaggtga agagacaaaa gctggaattg tgagcggata acaagctcaa 10560cacttgaaat ttaggaaaga gcagaatttg gcaaaaaaaa taaaaaaaaa ataaacacac 10620atactcatcg ag 10632930DNAunknownprimer 9ggagacauga attcaatgaa aatcgaagaa 301030DNAunknownprimer 10gggaaaguaa gcttaatcct tccctcgatc 301149DNAunknownprimer 11gactcatatg aaaatcgaag aaggtaaact ggtaatctgg attaacggc 491233DNAunknownprimer 12atataagctt tcaccttccc tcgatcccga ggt 331324DNAunknownprimer 13cttcaagggt caaccatcca aacc 241427DNAunknownprimer 14aatacgcgga tctttcacca actcttc 271527DNAunknownprimer 15gaagagttgg tgaaagatcc gcgtatt 271628DNAunknownprimer 16ctgagaattc tgaaatcctt ccctcgat 281724DNAunknownprimer 17ccagaagtga cggcaacggt gatt 241830DNAunknownprimer 18aagtgcggcg acgatagtca tgccccgcgc 301937DNAunknownprimer 19gtgatttctt atgacaacta cgtcaccatc cttgatg 372030DNAunknownprimer 20ttaaggatcc ttagtgcgcc tgatcccagt 302125DNAunknownprimer 21ccatagcata tgaaaatcga agaag 252227DNAunknownprimer 22cttgaatgca taacccccgt cagcagc 272330DNAunknownprimer 23ggttatgcat tcaagtatga aaacggcaag 302442DNAunknownprimer 24tatgcattca aatacggtga cattaaagac gtgggcgtgg at 422539DNAunknownprimer 25gcggcgtttt ccgcagtggc ggcaatacgt ggatctttc 392639DNAunknownprimer 26gcggaaaacg ccgcgaaagg tgaaatcatg ccgaacatc 392729DNAunknownprimer 27ggatggttgg ttcgctaaac tgcatcgtc 292829DNAunknownprimer 28tattaatcat tcttctcata tacttcaaa 292925DNAunknownprimer 29ggatggtgaa ggtcggtgtg aacgg 253028DNAunknownprimer 30tattactcct tggaggccat gtaggcca 283134DNAunknownprimer 31gcccaagctt gccaccatga aaatcgaaga aggt 343245DNAunknownprimer 32gcgctcgagc ttgtcatcgt catccgagct cgaattagtc tgcgc 453330DNAunknownprimer 33gcgctcgaga attccgcatt cggtagtatg 303443DNAunknownprimer 34ataagaatgc ggccgctcac gacgttgtaa aacgacggcc agt 43
Patent applications by Christopher H. Taron, Essex, MA US
Patent applications by Iris Walker, Rowley, MA US
Patent applications by Mehul Ganatra, Gloucester, MA US
Patent applications by Paul Riggs, Hooksett, NH US
Patent applications by Paul A. Colussi, Gloucester, MA US
Patent applications by Pei-Chung Hsieh, Topsfield, MA US
Patent applications by NEW ENGLAND BIOLABS, INC.
Patent applications in class Escherichia (e.g., E. coli, etc.)
Patent applications in all subclasses Escherichia (e.g., E. coli, etc.)