Patent application title: NUCLEIC ACIDS ENCODING RECOMBINANT PROTEIN A
Inventors:
James Ronald Peyser (Billerica, MA, US)
IPC8 Class: AC07K1600FI
USPC Class:
435 691
Class name: Chemistry: molecular biology and microbiology micro-organism, tissue cell culture or enzyme using process to synthesize a desired chemical compound or composition recombinant dna technique included in method of making a protein or polypeptide
Publication date: 2011-05-26
Patent application number: 20110124045
Abstract:
Disclosed are new recombinant nucleic acids encoding protein A
polypeptides and methods of using these nucleic acids.Claims:
1. An isolated nucleic acid molecule comprising a nucleic acid sequence
encoding a truncated protein A polypeptide that (i) includes some portion
of, but less than all of, a complete native X domain; (ii) lacks a signal
sequence; and (iii) binds specifically to an Fc region of an IgG
immunoglobulin.
2. The isolated nucleic acid molecule of claim 1, wherein the coding sequence is codon-optimized for expression in a non-pathogenic organism.
3. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid comprises a sequence at least 90% identical to SEQ ID NO:1 or SEQ ID NO:2.
4. The isolated nucleic acid molecule of claim 3, wherein the nucleic acid comprises a sequence at least 95% identical to SEQ ID NO:1 or SEQ ID NO:2.
5. The isolated nucleic acid molecule of claim 4, wherein the nucleic acid comprises SEQ ID NO:1 or SEQ ID NO:2.
6. The isolated nucleic acid molecule of claim 1, wherein the sequence encoding a truncated protein A polypeptide is operably linked to a bacterial ribosome binding site.
7. (canceled)
8. An expression vector comprising the nucleic acid molecule of claim 1 operably linked to an expression control sequence.
9. A bacterial cell comprising the vector of claim 8.
10. (canceled)
11. A bacterial cell transformed with the vector of claim 8, or a progeny of the cell, wherein the cell expresses a truncated protein A polypeptide.
12-13. (canceled)
14. A method of producing a truncated protein A polypeptide, the method comprising culturing the cell of claim 9 under conditions permitting expression of the polypeptide.
15. The method of claim 14, further comprising purifying the truncated protein A polypeptide from the cytoplasm of the cell.
16. A method of producing a truncated protein A polypeptide-containing affinity chromatography resin, said method comprising performing the method of claim 15 and immobilizing the truncated protein A polypeptide on a solid support material.
17-19. (canceled)
20. A method of purifying a protein comprising an Fc region of an IgG immunoglobulin, the method comprising contacting the protein A polypeptide-containing affinity chromatography resin made according to claim 16 with a solution comprising a protein comprising an Fc region of an IgG immunoglobulin; washing the substrate; and eluting bound protein comprising an Fc region of an IgG immunoglobulin.
21. An E. coli cell comprising an exogenous nucleic acid molecule that encodes a polypeptide consisting of SEQ ID NO:7.
22. The cell of claim 21, wherein the coding sequence is codon-optimized for expression in E. coli.
23. An isolated nucleic acid molecule that encodes a polypeptide comprising one or more nucleic acid sequences encoding a S. aureus protein A Ig-binding domain and a portion of a S. aureus protein A X-domain, wherein the nucleic acid sequence encoding the portion of the X-domain has a stop codon at position 379, 382, 385, 388, 391, 394, 397, 400, 403, 406, or 409 of the X domain coding sequence.
24. The nucleic acid of claim 23, wherein the one or more sequences encoding an Ig binding domain are wild-type.
25. The nucleic acid of claim 23, wherein the one or more sequence encoding an Ig binding domain are codon-optimized.
26. The nucleic acid of claim 23, wherein the sequence encoding the X domain is wild-type except for the stop codon.
27. The nucleic acid of claim 23, wherein the sequence encoding the X domain is codon-optimized.
28. (canceled)
Description:
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser. No. 11/952,082, filed on Dec. 6, 2007, which claims the benefit of prior U.S. Provisional Application No. 60/873,191, filed on Dec. 6, 2006. The disclosures of the prior applications are considered part of (and are incorporated by reference in) the disclosure of this application.
TECHNICAL FIELD
[0002] This invention relates to novel nucleic acids that encode truncated recombinant protein A polypeptides, vectors, cells, and methods of use.
BACKGROUND
[0003] Staphylococcal Protein A (SPA) is a protein that is found in nature anchored to the outer membrane of the gram-positive Staphylococcus aureus bacterium, the organism which is commonly associated with medically significant human "Staph" infections. The role of SPA in the life cycle of S. aureus remains uncertain, but some studies have correlated the presence of SPA with pathogenicity of the organism.
[0004] Functionally, SPA is well known for its ability to tightly, but reversibly, bind to the constant region of an immunoglobulin molecule (IgG). This property has been widely exploited in the affinity purification of antibodies for commercial uses. For example, SPA can be purified from S. aureus and covalently bound to various forms of solid supports to thus immobilize it to make an affinity chromatography resin. Crude preparations of antibodies can then be passed over such an immobilized SPA resin to bind and capture the commercially valuable antibody, while contaminating materials are washed away. The bound antibody may then be eluted in pure form by a simple adjustment of the pH.
SUMMARY
[0005] The invention is based, at least in part, on new recombinant nucleic acid sequences encoding truncated versions of protein A polypeptides (e.g., rSPA) that (i) include some portion (but not all) of the X-domain of native protein A, (ii) do not include a signal sequence and (iii) bind specifically to an Fc region of an IgG immunoglobulin. The new nucleic acids have the advantage of being suitable for use in efficiently expressing a truncated form of protein A polypeptides in non-pathogenic bacteria, especially E. coli, without being significantly degraded within the bacteria. Thus, the nucleic acids described herein can be used in laboratory and/or manufacturing practices that do not require a pathogenic S. aureus host for the production of protein A polypeptides. The truncated rSPA that is encoded by said the new nucleic acid sequences has the useful advantage that it contains some portion of the X domain, which portion significantly improves its ability to be immobilized for use as an affinity chromatography reagent. A means of efficiently producing a form of rSPA that contains some portion of the X domain in E. coli or other non-pathogenic bacteria, has not previously been disclosed.
[0006] In one aspect, the invention features isolated nucleic acid molecules that include a nucleic acid sequence encoding truncated Staphylococcus aureus protein A polypeptides. The protein A polypeptides have one or more of the following features: (i) includes less than a complete native X domain; (ii) does not include a signal sequence (e.g., the nucleotide does not encode a signal sequence) or a heterologous N-terminal sequence; (iii) binds specifically to an Fc region of an IgG immunoglobulin; (iv) is not substantially degraded when expressed in a heterologous host (e.g., a non-Staphylococcal host such as E. coli); and (v) includes only Staphylococcal polypeptide sequences. The coding sequence can be codon-optimized for expression in a non-pathogenic organism (e.g., E. coli). In some embodiments, the nucleic acid includes a sequence at least 80% identical (e.g., at least 85%, 90%, 95%, 98%, 99%, or 100% identical) to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:22. The nucleic acid sequence can be operably linked to a bacterial ribosome binding site, e.g., ACGCGTGGAGGATGATTAA (SEQ ID NO:3). In some embodiments, the protein A polypeptides bind to the Fc region of human IgG1 with an affinity of 1000 nM or less (e.g., 500 nM or less, 200 nM or less, 50 nM or less, 20 nM or less, 10 nM or less, or 5 nM or less) in 0.02 M sodium phosphate, pH 7.0.
[0007] The invention also features isolated nucleic acid molecules that encode a polypeptide, which include one or more nucleic acid sequences encoding an S. aureus protein A Ig-binding domain and a portion of an S. aureus protein A X-domain, wherein the nucleic acid sequence encoding the portion of the X-domain has a stop codon at position 379, 382, 385, 388, 391, 394, 397, 400, 403, 406, or 409 of the X domain coding sequence. In some embodiments, the one or more sequences encoding an Ig binding domain are wild-type. In other embodiments, the one or more sequence encoding an Ig binding domain are codon-optimized. In some embodiments, the sequence encoding the X domain is "wild-type" except for the stop codon. In other embodiments, the sequence encoding the X domain is codon-optimized. In some embodiments, the polypeptide sequence contains only amino acid sequences found in a native Staphylococcus derived protein A.
[0008] In another aspect, the invention features vectors that include any of the nucleic acid molecules described herein. The vectors can be expression vectors, wherein the polypeptide-encoding nucleic acid sequences are operably linked to expression control sequences (e.g., a promoter, activator, or repressor). The invention also features bacterial cells, e.g., non-pathogenic bacterial cells (e.g., E. coli), that include the above vectors and bacterial cells that include polypeptide-encoding nucleic acid sequences described herein operably linked to an expression control sequence. In other embodiments, the invention also features bacterial cells, e.g., non-pathogenic bacterial cells (e.g., E. coli) transformed with the above vectors, and the progeny of such cells, wherein the cells express a truncated protein A or a polypeptide that includes a protein A Ig-binding domain and a portion of a protein A X domain.
[0009] The invention also features E. coli cells that include an exogenous nucleic acid molecule that encodes a polypeptide that includes SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:17. In some embodiments, the nucleic acid sequence that encodes the polypeptide is codon-optimized for expression in E. coli. In some embodiments, the nucleic acid sequence includes SEQ ID NO:1 or SEQ ID NO:22. In some embodiments, the protein A binds to the Fc region of human IgG1 (e.g., with an affinity of 1000 nM or less (e.g., 500 nM or less, 200 nM or less, 50 nM or less, 20 nM or less, 10 nM or less, or 5 nM or less)) in 0.02 M sodium phosphate, pH 7.0.
[0010] In other embodiments, the invention features methods of producing truncated protein A polypeptides that include one or more protein A Ig-binding domains and a portion of a protein A X domain. The methods include culturing any of the cells described herein under conditions permitting expression of the polypeptide. The methods can further include purifying the protein A polypeptide from the cytoplasm of the cell. In some embodiments, the protein A polypeptide is then immobilized on a solid support material, e.g., cellulose, agarose, nylon, or silica. In some embodiments, the solid substrate is a porous bead, a coated particle, or a controlled pore glass. The invention also features solid support materials on which the protein A polypeptide has been immobilized.
[0011] The invention also features methods of purifying a protein A polypeptide that includes an Fc region of an IgG immunoglobulin. The methods include contacting the truncated protein A polypeptide-bound substrate made as described herein with a solution that includes a protein that includes an Fc region of an IgG immunoglobulin; washing the substrate; and eluting bound a polypeptide that includes an Fc region of an IgG immunoglobulin. The invention also features protein A polypeptides (e.g., proteins that include an Fc region of an IgG immunoglobulin) purified by the methods described herein or using solid support materials described herein.
[0012] As used herein, "truncated protein A polypeptide" refers to a protein A polypeptide that (i) includes some, but not all, of a native X-domain, and (ii) binds specifically to an Fc region of an IgG immunoglobulin.
[0013] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
[0014] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
DESCRIPTION OF DRAWINGS
[0015] FIG. 1 is a schematic map of native protein A domains.
[0016] FIG. 2 is an amino acid sequence (SEQ ID NO:4) of native Staphylococcus aureus (strain 8325-4) protein A (Lofdahl et al., Proc. Natl. Acad. Sci. USA, 80:697-701, 1983). N-terminal underlined sequence represents S. aureus signal peptide. C-terminal underlined sequence represents the X-domain.
[0017] FIG. 3 is an example of a protein A amino acid sequence broken into the designated domains: IgG binding E domain (SEQ ID NO:9), IgG binding D domain (SEQ ID NO:10), IgG binding A domain (SEQ ID NO:11), IgG binding B domain (SEQ ID NO:12), IgG binding C domain (SEQ ID NO:13), X domain (X domain 1)(SEQ ID NO:14) and example of portion of X domain used to make recombinant protein shown in FIG. 6 (X domain 2)(SEQ ID NO:15).
[0018] FIG. 4 is an amino acid sequence (SEQ ID NO:7) of an exemplary truncated protein A lacking portions of the X domain as seen in FIG. 3 as bolded amino acids. The sequences underlined in SEQ ID NO:7 are repetitive eight amino acid sequences (KPGKEDXX); SEQ ID NO:8).
[0019] FIG. 5 is a second example of an amino acid sequence (SEQ ID NO:6) of a recombinant S. aureus protein A polypeptide with a portion of the X domain.
[0020] FIG. 6 is a plasmid map of pREV2.1-rSPA containing genetic elements for expression of the rSPA recombinant gene. Sequence landmarks are noted and include the β-glucuronidase promoter, ribosome binding site (RBS), multiple cloning site (MCS) and Trp terminator. The plasmid backbone is defined as the ˜3900 by DNA sequence between the MluI and BamHI restriction sites.
[0021] FIG. 7 is a partial nucleotide sequence (SEQ ID NO:16) of an E. coli expression vector. The nucleotide sequence includes a portion of the vector backbone at 5' and 3' terminal sequences (italics), the promoter sequence is underlined, and the start methionine and the termination codon are in bold.
[0022] FIG. 8 is a depiction of an immunoblot using antibodies that bind specifically to recombinant protein A polypeptides produced in E. coli cells. Lane 1: rPA50; Lane 2: vector control; Lane 3; clone 7a; Lane 4: clone 9a; Lane 5: clone 10a; Lane 6: clone 19a.
[0023] FIGS. 9A-B are graphs comparing the results of dynamic binding capacity experiments using (i) truncated protein A polypeptide produced using a nucleic acid described herein and (ii) PROSEP® A chromatography media (Millipore) as a commercially available comparison.
[0024] FIG. 10 is an exemplary nucleotide sequence (SEQ ID NO:2) that encodes a truncated protein A polypeptide.
[0025] FIG. 11 is an amino acid sequence (SEQ ID NO:17) of an exemplary truncated protein A polypeptide lacking a portion of the X domain.
[0026] FIG. 12 is an amino acid sequence (SEQ ID NO:5) of an exemplary truncated protein A polypeptide lacking a portion of the X domain.
[0027] FIG. 13 is an exemplary nucleic acid sequence (SEQ ID NO:22) encoding a truncated protein A polypeptide.
DETAILED DESCRIPTION
[0028] Described herein are novel nucleic acids and methods for the expression of truncated forms of protein A that include some portion, but less than all, of the native X-domain, only polypeptide sequences found in native S. aureus protein A, and bind specifically to IgG immunoglobulin Fc region. The truncated forms of protein A can be expressed cytoplasmically (e.g., without a signal peptide) and harvested from a non-pathogenic host, for example, non-pathogenic strains of E. coli, which are generally considered safer to handle and use than S. aureus. Furthermore, molecular biological and fermentation techniques for E. coli have been developed that permit high levels of truncated protein A expression and recovery.
Structure of Full Length Protein A Precursor
[0029] SPA is a cell surface protein that can be isolated from particular strains of Staphylococcus aureus. The protein is able to bind free IgG and IgG-complexes. Membrane-bound protein A has been identified in the following S. aureus strains: NCTC 8325-4 (Iordanescu and Surdeanu, J. Gen. Microbiol., 96:277-281, 1976), NCTC 8530, i.e., Cowanl or ATCC 12598; and SA113 or ATCC 35556. A soluble form of protein A is expressed by S. aureus strain A676 (Lindmark et al., Eur. J. Biochem., 74:623-628, 1977). The ATCC strains described herein, as well as other S. aureus strains, are available from American Tissue Culture Collection (Bethesda, Md.).
[0030] The gene encoding the full length SPA precursor is known as spa. Nucleotide and protein sequences for spa are publicly available, e.g., through GENBANK nucleotide database at Accession No. J01786 (complete coding sequence) and/or BX571856.1 (genomic sequence of clinical S. aureus strain that includes coding sequence for GENPEPT Accession No. CAG39140). See also, U.S. Pat. No. 5,151,350. In spite of the various sequences available to the public, the inventors believe that the new nucleic acid sequences described herein have not been previously isolated, sequenced, or publicly described.
[0031] Structurally, the SPA protein consists of an amino-terminal signal peptide followed by five highly homologous immunoglobulin binding domains and a so-called X domain (see FIG. 1). The signal peptide directs the SPA protein for secretion through the membrane and is thereafter removed by proteolysis. The five immunoglobulin binding domains, named A through E, are arranged as E-D-A-B-C in most naturally occurring forms of the molecule. The X domain, which lies at the carboxy terminus and is believed to be involved in anchoring the SPA to and extending it from the outer membrane of the bacterium, consists of two structurally distinct regions, the first of which comprises a series of highly repetitive blocks of octapeptide sequence (termed Xr) and the second of which is a hydrophobic region at the extreme C-terminus (termed Xc), which is thought to anchor the SPA molecule into the cell membrane. The entire SPA molecule thus consists of seven distinct domains that are structurally arranged as [S]-E-D-A-B-C-X.
[0032] A number of strains of S. aureus are known and the protein sequence of the SPA from several of these has been reported in the prior art. A comparison of these SPA sequences reveals a significant amount of genetic variability from one strain to another, which can include point mutations, domain deletions, repetitive sequence insertions, and genetic rearrangements. The effect of such differences on SPA function has not been well studied, although it appears that deletion of at least a portion of the Xc domain results in a form of SPA that is secreted into the culture medium (Lindmark et al., Eur. J. Biochem., 74:623-628, 1977).
[0033] The IgG Fc region-binding domains of S. aureus include highly repetitive sequences at the protein level and, to a lesser extent, at the nucleic acid sequence level. Strain 8325-4 produces protein A that includes five IgG-binding domains that are schematically represented in FIG. 1 as regions E, D, A, B, and C. These domains bind specifically to the Fc and/or Fab portion of IgG immunoglobulins to at least partly inactivate an S. aureus-infected host's antibodies. By binding to the Fc region of immunoglobulins, protein A inhibits binding of IgGs to complement and Fc receptors on phagocytic cells, thus blocking complement activation and opsonization.
[0034] The X domain is a C-terminal region that contains (i) Xr, a repetitive region with approximately twelve repetitive eight amino acid sequences and (ii) Xc, an approximately 80 to 95 amino acid constant region at the C-terminus of the protein. Each repetitive amino acid sequence generally includes a KPGKEDXX (SEQ ID NO:8) motif, wherein in some embodiments the XX dipeptide can be NN, GN, or NK. See e.g., Uhlen et al., J. Biol. Chem. 259:1695-1702, 1984, and underlined residues in FIG. 5. The X domain is involved in the targeting and anchoring protein A to the cell surface of S. aureus.
[0035] Although the X domain is not involved in IgG binding, it may be useful to retain a portion of the X domain (e.g., when expression protein A polypeptide by recombinant means) for the purpose of improving the properties of the rSPA in the preparation of an affinity chromatography resin. For example, a portion of the X domain can serve as a "molecular stalk" to tether the IgG-binding regions of the polypeptide to a solid substrate. Moreover, a portion of the X domain can act to present the IgG-binding regions of the polypeptide at a distance out and away from a solid substrate to which it is tethered in order to better allow interactions of the IgG-binding regions to Fc-containing polypeptides. Further, the inclusion of a portion of the X domain can potentially improve folding and/or stability of the protein A molecule over the folding and/or stability of the protein A molecule without the X domain. Finally, certain of the amino acid side chains, e.g., lysine, present in the X domain can provide convenient reaction sites to enable efficient covalent coupling to a solid support without compromising the functional properties of the IgG binding domains.
[0036] The signal peptide (SP) is an N-terminal extension present in proteins destined for export by the general (Sec-dependent) bacterial secretion system. SP mediates recognition of the nascent unfolded polypeptide chain by the Sec-dependent secretion apparatus, translocation through the cell membrane, and cleavage by the signal peptidase (reviewed by van Wely et al., FEMS Microbiol. Rev., 25:437-54, 2001). Secretion is sometimes necessary to achieve stable polypeptide expression. Cytoplasmic expression of recombinant proteins may fail because of toxicity of the protein, a requirement of the secretion process for proper folding of the protein, or instability of the protein in the cytoplasmic environment. Stable recombinant protein expression can sometimes achieved by enclosing the polypeptide sequence of interest with flanking regions of heterologous amino acids.
[0037] While it may be desirable to express an rSPA that contains at least a portion of the X domain, no demonstration of such a protein being produced free of heterologous sequences has been reported. Attempts to produce a recombinant protein A containing a portion of the X domain by secretion in E. coli produced a protein product that was extensively degraded by endogenous proteases (Uhlen et al., J. Bacteriol., 159:713-719, 1984). Another challenge that has been noted in attempting to express a full-length rSPA gene product in E. coli is that the Xc region can be toxic to the cells (Warnes et al., Curr. Microbiol., 26:337-344, 1993). These findings have led at least some investigators to eliminate the X domain when expressing the rSPA gene in E. coli (see, e.g., Hellebust et al., J. Bacteriol. 172:5030-34, 1990). The new sequences and systems described herein provide for high levels of expression in E. coli of proteolytically stable forms of rSPA that contain a portion of the X domain. These X domain containing forms of rSPA have particular utility in the creation of rSPA containing affinity chromatography resins.
Virulence of S. Aureus
[0038] The Center for Disease Control and World Health Organization classify S. aureus a Biosafety Level II or Group II infectious agent, respectively. These classifications are reserved for agents associated with human disease and hazards of percutaneous injury, ingestion, and/or mucous membrane exposure.
[0039] S. aureus is a major cause of hospital-acquired (nosocomial) infections associated with surgical wounds and implanted medical devices. This bacterium can release enterotoxins responsible for food poisoning, and superantigens released by S. aureus can induce toxic shock syndrome. S. aureus also causes a variety of suppurative (pus-forming) infections and toxinoses in humans, as well as skin lesions including boils, styes, and furunculosis. S. aureus has also been found to co-infect subjects with pneumonia, mastitis, phlebitis, meningitis, urinary tract infections, and deep-seated infections, such as osteomyelitis and endocarditis.
[0040] S. aureus expresses a number of potential virulence factors: (1) surface proteins that promote colonization of host tissues; (2) invasins (e.g., leukocidin, kinases, hyaluronidase) that promote bacterial spread in tissues; (3) surface factors (e.g., capsule, protein A) that inhibit phagocytic engulfment; (4) biochemical properties that enhance their survival in phagocytes (carotenoids, catalase production); (5) immunological disguises (protein A, coagulase, clotting factor); (6) membrane-damaging toxins that lyse eukaryotic cell membranes (hemolysins, leukotoxin, and leukocidin); (7) exotoxins that damage host tissues or otherwise provoke symptoms of disease (staphylococcal enterotoxins (SE) A-G, toxic shock syndrome toxin (TSST), exfoliative toxin (ET)); and (8) inherent and acquired resistance to antimicrobial agents.
[0041] Thus, the virulence level of S. aureus is more severe than that for Biosafety Level 1 or Group 1 organisms, such as laboratory and commercial strains of E. coli. Biosafety Level 1 is reserved for well-characterized organisms not known to cause disease in healthy adult humans, and of minimal potential hazard to laboratory personnel and the environment.
Nucleic Acids Encoding Truncated Protein A Polypeptides
[0042] In one aspect, described herein are certain nucleic acids encoding a truncated protein A polypeptide that has one or more of the following characteristics: (i) contains only sequences coding for SPA, i.e., does not contain heterologous sequences, (ii) includes some portion of, but less than all of, the complete native X domain, (iii) binds specifically to an IgG immunoglobulin Fc region, and (iv) lacks a signal sequence. Exemplary nucleic acids include, but are not limited to, nucleic acids encoding SEQ ID NO:1 and variants thereof that have been codon optimized for expression in a specific host such as E. coli.
[0043] An exemplary nucleic acid encoding a truncated S. aureus protein A polypeptide is as follows.
TABLE-US-00001 (SEQ ID NO: 1) ATGGCGCAACACGATGAAGCTCAACAGAACGCTTTTTACCAGGTACT GAACATGCCGAACCTGAACGCGGATCAGCGCAACGGTTTCATCCAGA GCCTGAAAGACGACCCTTCTCAGTCCGCAAACGTTCTGGGCGAGGCT CAGAAACTGAACGACAGCCAGGCCCCAAAAGCAGATGCTCAGCAAAA TAACTTCAACAAGGACCAGCAGAGCGCATTCTACGAAATCCTGAACA TGCCAAATCTGAACGAAGCTCAACGCAACGGCTTCATTCAGTCTCTG AAAGACGATCCGTCCCAGTCCACTAACGTTCTGGGTGAAGCTAAGAA GCTGAACGAATCCCAGGCACCAAAAGCAGACAACAACTTCAACAAAG AGCAGCAGAACGCTTTCTATGAAATCTTGAACATGCCTAACCTGAAT GAAGAACAGCGTAACGGCTTCATCCAGTCTCTGAAGGACGACCCTAG CCAGTCTGCTAACCTGCTGTCCGAAGCAAAAAAACTGAACGAGTCCC AGGCTCCAAAAGCGGATAACAAATTCAACAAGGAGCAGCAGAACGCA TTCTACGAAATCCTGCACCTGCCGAACCTGAACGAAGAACAGCGTAA CGGTTTCATCCAATCCCTGAAAGACGATCCTTCCCAGTCCGCAAATC TGCTGGCAGAAGCAAAGAAACTGAACGACGCACAGGCACCGAAGGCT GACAACAAGTTCAACAAAGAGCAGCAGAATGCCTTCTACGAGATTCT GCATCTGCCAAACCTGACTGAGGAGCAGCGCAACGGTTTCATTCAGT CCCTGAAGGACGACCCAAGCGTCAGCAAGGAAATCCTGGCTGAGGCG AAAAAACTGAACGATGCACAGGCTCCGAAGGAAGAAGACAACAATAA ACCTGGTAAAGAAGATAATAATAAGCCTGGCAAGGAAGATAACAACA AGCCGGGCAAGGAGGACAACAATAAACCGGGCAAAGAGGATAATAAC AAGCCTGGTAAGGAAGACAACAACAAACCAGGCAAAGAAGATGGCAA CAAGCCGGGTAAGGAGGATAATAAAAAACCAGGCAAGGAAGACGGCA ACAAACCTGGCAAGGAGGATAACAAAAAGCCAGGCAAGGAGGATGGT AATAAACCGGGCAAAGAAGACGGCAACAAGCCTGGTAAAGAAGACGG TAACGGTGTACACGTCGTTAAACCTGGTGACACCGTGAACGACATCG CTAAGGCTAATGGCACCACGGCAGACAAGATTGCAGCGGACAATAAA TAA
[0044] For both SEQ ID NO:1 and SEQ ID NO:22, the E domain is encoded by nucleotides 2-171; the D domain is encoded by nucleotides 172-354; the A domain is encoded by nucleotides 355-528; the B domain is encoded by nucleotides 529-702; the C domain is encoded by nucleotides 703-876; and the X domain is encoded by nucleotides 877-1272.
[0045] Certain genes can provide challenges for efficient expression by recombinant means in heterologous hosts. Alteration of the codons native to the sequence can facilitate more robust expression of these proteins. Codon preferences for abundantly expressed proteins have been determined in a number of species, and can provide guidelines for codon substitution. Synthesis of codon-optimized sequences can be achieved by substitution of codons in cloned sequences, e.g., by site-directed mutagenesis, or by construction of oligonucleotides corresponding to the optimized sequence by chemical synthesis. See, e.g., Mirzabekov et al., J. Biol. Chem., 274:28745-50, 1999.
[0046] The optimization should also include consideration of other factors such as the efficiency with which the sequence can be synthesized in vitro (e.g., as oligonucleotide segments) and the presence of other features that affect expression of the nucleic acid in a cell. For example, sequences that result in RNAs predicted to have a high degree of secondary structure should be avoided. AT- and GC-rich sequences that interfere with DNA synthesis should also be avoided. Other motifs that can be detrimental to expression include internal TATA boxes, chi-sites, ribosomal entry sites, prokaryotic inhibitory motifs, cryptic splice donor and acceptor sites, and branch points. These features can be identified manually or by computer software and they can be excluded from the optimized sequences.
[0047] Nucleic acids described herein include recombinant DNA and synthetic (e.g., chemically synthesized) DNA. Nucleic acids also include recombinant RNAs, e.g., RNAs transcribed (in vitro or in vivo) from the recombinant DNA described herein, or synthetic (e.g., chemically synthesized) RNA.
[0048] Nucleic acids can be double-stranded or single-stranded. Where single-stranded, the nucleic acid can be a sense strand or an antisense strand. Nucleic acids can be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such oligonucleotides can be used, for example, to prepare nucleic acids that have increased resistance to nucleases.
[0049] The term "purified," referring, e.g., to a polypeptide, denotes a molecule that is substantially free of cellular or viral material with which it is naturally associated or recombinantly expressed, or chemical precursors or other chemicals used for chemical synthesis.
[0050] Also described herein are variants of nucleic acids encoding truncated rSPA molecules. Such variants code for IgG-binding, truncated versions of protein A polypeptides that (i) include a portion of but less than the complete X domain of SPA, (ii) are suitable for expression in E. coli, and (iii) are substantially identical to SEQ ID NO:6 or SEQ ID NO:7. In some embodiments, the nucleic acids do not encode a signal sequence. A variant nucleic acid (e.g., a codon-optimized nucleic acid) encoding a truncated protein A molecule can be substantially identical, i.e., at least 75% identical, e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical, to SEQ ID NO:1 or SEQ ID NO:22. In certain embodiments, a truncated rSPA variant that is "substantially identical" to SEQ ID NO:6 or SEQ ID NO:7 is a polypeptide that is at least 75% identical (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical) to a SEQ ID NO:6 or SEQ ID NO:7.
[0051] The determination of percent identity between two nucleotide or polypeptide sequences can be accomplished using the BLAST 2.0 program, which is available to the public at ncbi.nlm.nih.gov/BLAST. Sequence comparison is performed using an ungapped alignment and using the default parameters (gap existence cost of 11, per residue gap cost of 1, and a lambda ratio of 0.85). When polypeptide sequences are compared, a BLOSUM 62 matrix is used. The mathematical algorithm used in BLAST programs is described in Altschul et al., 1997, Nucleic Acids Research, 25:3389-3402.
[0052] Nucleic acid variants of a sequence that contains SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:22 include nucleic acids with a substitution, variation, modification, replacement, deletion, and/or addition of one or more nucleotides (e.g., 2, 3, 4, 5, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100 nucleotides) from a sequence that contains SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:22. All of the aforementioned nucleic acid variants encode a recombinant truncated polypeptide that (i) is suitable for expression in a non-pathogenic, heterologous host cell, (ii) contains a portion of, but less than all of, the complete X-domain of SPA, and (iii) specifically binds to IgG. In particular, the term "variant" covers nucleotide sequences encoding polypeptides that are capable of binding to IgG through introduction of additional S. aureus protein A derived polypeptide sequences, for example, from additional strains of S. aureus.
Vectors, Plasmids, and Host Cells
[0053] Nucleic acids encoding a truncated rSPA polypeptide as described herein can be operably linked to genetic constructs, e.g., vectors and plasmids. In some cases a nucleic acid described herein is operably linked to a transcription and/or translation sequence in an expression vector to enable expression of a truncated rSPA polypeptide. By "operably linked," it is meant that a selected nucleic acid, e.g., a coding sequence, is positioned such that it has an effect on, e.g., is located adjacent to, one or more sequence elements, e.g., a promoter and/or ribosome binding site, which directs transcription and/or translation of the sequence.
[0054] Some sequence elements can be controlled such that transcription and/or translation of the selected nucleic acid can be selectively induced. Exemplary sequence elements include inducible promoters such as tac, T7, PBAD (araBAD), and β-D-glucuronidase (uidA) promoter-based vectors. Control of inducible promoters in E. coli can be enhanced by operably linking the promoter to a repressor element such as the lac operon repressor (lacR). In the specific case of a repressor element, "operably linked" means that a selected promoter sequence is positioned near enough to the repressor element that the repressor inhibits transcription from the promoter (under repressive conditions).
[0055] Typically, expression plasmids and vectors include a selectable marker (e.g., antibiotic resistance gene such as TetR or AmpR). Selectable markers are useful for selecting host cell transformants that contain a vector or plasmid. Selectable markers can also be used to maintain (e.g., at a high copy number) a vector or plasmid in a host cell. Commonly used bacterial host plasmids include pUC series of plasmids and commercially available vectors, e.g., pAT153, pBR, PBLUESCRIPT, pBS, pGEM, pCAT, pEX, pT7, pMSG, pXT, pEMBL. Another exemplary plasmid is pREV2.1.
[0056] Plasmids that include a nucleic acid described herein can be transfected or transformed into host cells for expression of truncated rSPA polypeptides. Techniques for transfection and transformation are known in the art, including calcium phosphatase transformation and electroporation. In certain embodiments, transformed host cells include non-pathogenic prokaryotes capable of highly expressing recombinant proteins. Exemplary prokaryotic host cells include laboratory and/or industrial strains of E. coli cells, such as BL21 or K12-derived strains (e.g., C600, DH1α, DH5α, HB101, INV1, JM109, TB1, TG1, and X-1Blue). Such strains are available from the ATCC or from commercial vendors such as BD Biosciences Clontech (Palo Alto, Calif.) and Stratagene (La Jolla, Calif.). For detailed descriptions of nucleic acid manipulation techniques, see Ausubel et al., eds., Current Protocols in Molecular Biology, Wiley Interscience, 2006, and Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, 2001.
Expression and Purification of Truncated Protein A Polypeptides
[0057] Host cells containing a nucleic acid encoding a truncated rSPA can be grown under conditions suitable for expression of said encoded truncated rSPA. Host cells can be grown that constitutively express truncated rSPA. In other systems, host cells are first grown under conditions that inhibit expression of truncated rSPA and are later switched to media that induces expression of truncated rSPA, for example, by activating or derepressing promoter operably linked to the rSPA coding sequence.
[0058] In another exemplary system, a bacterial host cell includes the coding sequence for a truncated rSPA (operably linked to T7 promoter), a T7 RNA polymerase (operably linked to lac operon/lac promoter control region), and a lac repressor (lacI gene). The lac repressor can bind to the lac operon and prevent bacterial RNA polymerase binding to the lac promoter region, thereby inhibiting T7 polymerase expression. Bacterial host cells can be cultured, e.g., in fermentation tanks When the host culture reaches a desired population density (e.g., population reaches exponential or "log" growth), isopropyl-beta-D-thiogalactopyranoside (IPTG) is added to the bacterial growth media. IPTG binds to and inactivates the lac repressor, thereby derepressing the lac operon/lac promoter and allowing expression of T7 polymerase. T7 polymerase expression, in turn, can drive high level expression of truncated rSPA.
[0059] After host cells have been grown under conditions suitable for expression of truncated rSPA, host cells are harvested and rSPA protein is purified from other host cell material. Typically, host cells are lysed in the presence of protease inhibitors and truncated rSPA is separated from cell debris, e.g., by low speed centrifugation. Further enrichment of rSPA material is optionally accomplished by serial centrifugations and isolation of fractions containing rSPA.
[0060] In certain embodiments, purification of truncated rSPA includes binding to purification media such as a resin or magnetic beads. In these embodiments, purification media includes IgG, or fragments thereof, that bind to protein A. IgG fragments that bind to protein A include Fc or Fab fragments. In other embodiments, purification media includes nickel-nitrilotriacetic acid (Ni-NTA), maltose, glutathione, or any other material that binds to a truncated rSPA fusion protein. After binding of truncated rSPA to purification media, the purification media is washed, e.g., with a salt buffer or water, and truncated rSPA is eluted from the purification media with an elution buffer. Elution buffer includes a composition that disrupts truncated rSPA binding to the purification media. For example, elution buffers can include glycine to disrupt IgG-truncated protein A interactions, imidazole or urea to disrupt His-tag-Ni-NTA interactions, and/or glutathione to disrupt GST-glutathione interactions. Truncated rSPA is recovered by batch or column elution.
[0061] Eluted rSPA can be further purified using chromatography techniques, e.g., ion exchange chromatography, affinity chromatography, gel filtration (or size exclusion) chromatography. In addition, purified truncated rSPA can be concentrated by binding a solution of purified rSPA to purification media and subsequently eluting bound truncated rSPA in a smaller volume of elution buffer.
[0062] For detailed protein purification techniques, see Scopes, Protein Purification: Principles and Practice, Springer Science, NY, 1994.
Substrates
[0063] Described herein are new methods of making useful resins and other substrates to which truncated rSPA can be attached. Generally, a nucleic acid described herein is used to express truncated rSPA, which is purified, and subsequently attached to a substrate. Substrates can include organic and inorganic materials. Substrates can be manufactured in useful forms such as microplates, fibers, beads, films, plates, particles, strands, gels, tubing, spheres, capillaries, pads, slices, or slides. Substrate material can include, for example, magnetic beads, porous beads (e.g., controlled pore glass beads), cellulose, agarose (e.g., SEPHAROSE®), coated particles, glass, nylon, nitrocellulose, and silica.
[0064] In some embodiments, truncated rSPA is expressed in a non-pathogenic host from a nucleic acid described herein, the rSPA is purified from host material, and the rSPA is attached (e.g., covalently attached) to a porous substrate that is hydrophobic and/or protein absorptive. Such substrates or supports include ion exchange packings and bioaffinity packings
[0065] In certain embodiments, truncated rSPA is harvested and purified as described herein from a non-pathogenic organism, and the rSPA is attached to a porous protein-adsorptive support having hydroxyl groups on its surface. Exemplary supports include porous metalloid oxides, porous metallic oxides, and porous mixed metallic oxides. Such materials include silica, e.g., silica particles or silica gel, alumina, stannia, titania, zirconia, and the like. In some embodiments, the porous support has a particle size of about 0.5 to about 800 micrometers, e.g., about 5 to about 60 micrometers, and a pore diameter of about 30 to about 300 angstroms, e.g., about 60 angstroms.
[0066] 1. Exemplary Substrates--Porous Silica (Including Controlled Pore Glass)
[0067] Porous silica, including controlled pore glass, has been described in U.S. Pat. Nos. 3,549,524 and 3,758,284 and is commercially available from vendors such as Prime Synthesis, Inc. (Aston, Pa.) and Millipore (Billerica, Mass.). Porous silica supports may undergo various treatments prior to being attached to truncated protein A polypeptides. Generally, a silica support is derivatized to introduce reactive functional groups. The derivatized support is activated and then coupled to truncated rSPA produced by the methods described herein.
[0068] For example, silica supports can be derivatized using an arginine-containing linker as described in U.S. Pat. No. 5,260,373. Silica supports can be amino-derivatized by a silanization process, e.g., using aminosilanes such as γ-aminopropyltrimethoxysilane 6-(aminohexylaminopropyl)trimethoxy silane, aminoundecyltrimethoxysilane, p-aminophenyltrimethoxysilane, 4-aminobutyltrimethoxysilane, and (aminotheylaminoethyl)-phenyltrimethoxysilane. Dual zone silanization can be employed, e.g., as described in U.S. Pat. Nos. 4,773,994, 4,778,600, 4,782,040, 4,950,634, and 4,950,635. Silica supports can also be amino-derivatized using o-dianisidine, e.g., as described in U.S. Pat. No. 3,983,000. Amino-derivatized supports can be carboxy-derivatized by a second reaction with, e.g., succinic anhydride, e.g., as described in U.S. Pat. No. 4,681,870. Amino-derivatized supports can also be treated with an aldehydes, e.g., gluteraldehyde, to introduce reactive aldehyde groups, as described in U.S. Pat. Nos. 3,983,000 and 4,681,870. Derivatized porous silica can also be obtained commercially from Prime Synthesis, Inc.
[0069] Derivatized porous silica can be activated and reacted and bound to truncated rSPA in aqueous solution. For example, aqueous peptide solutions react directly with o-dianisidine and/or gluteraldehyde coated substrates. In other examples, carbodiimide and rSPA are mixed with derivatized substrate, such that carbodiimide reacts with and attaches rSPA to the derivatized substrate, e.g., as described in U.S. Pat. No. 4,681,870.
[0070] Other methods for attaching a peptide to porous silica can also be used in the methods described.
[0071] 2. Exemplary Substrates--Agarose
[0072] A variety of agarose substrates known in the art can also be used in the methods described herein. For example, agarose substrates (e.g., cross-linked, beaded agarose) suitable for use in chromatography packing resin can be derivatized, activated, and linked to a truncated rSPA produced according to the methods described herein.
[0073] Derivatized agarose can be manufactured using methods known in the art. For example, agarose can be derivatized and activated using an arginine-containing linker as described in U.S. Pat. No. 5,260,373. Activated and derivatized agarose products suitable for peptide linking are also commercially available from manufacturers such as Amersham Biosciences (Piscataway, N.J.). These include N-hydroxysuccinimide (NHS)-activated SEPHAROSE® 4 FAST FLOW designed for the covalent coupling through the primary amine of a ligand, CNBr-activated SEPHAROSE® designed for the attachment of larger primary amine containing ligands under mild conditions, EAH Sepharose 4B designed for coupling of small ligands containing free carboxyl groups via a 10-atom spacer arm using carbodiimide as the coupling, and ECH Sepharose 4B for coupling small ligands containing free amino groups via a 9-atom spacer arm also using carbodiimide as the coupling reagent. Instructions for coupling derivatized SEPHAROSE® to peptides can be obtained from the manufacturer.
[0074] Generally, truncated rSPA can be coupled to derivatized agarose substrates by incubating rSPA and the activated substrate in an aqueous solution. Coupling conditions can include salt buffers such as 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), sodium carbonate, sodium chloride, potassium phosphate and other salts. Carbodiimide can also be used as needed or desired.
Applications For Truncated rSPA
[0075] Nucleic acids described herein are useful for the cost effective, efficient, and less hazardous production of truncated rSPA in non-pathogenic hosts such as E. coli as compared to harvesting of similar peptides from S. aureus. The rSPA produced by the nucleic acids described herein can be covalently linked to substrates with greater efficiency than forms of rSPA that are lacking the X domain.
[0076] Truncated rSPA can be also used in a wide array of industries including research, medical diagnostics, and the discovery and manufacture of therapeutic biologics. Research applications include use as a reagent in immunoprecipitation and antibody purification protocols. Truncated rSPA can be used as a component in diagnostic tools that isolate or evaluate antibodies in an organism.
[0077] Truncated rSPA is particularly useful for the manufacture of affinity chromatography resins that are widely used for large-scale purification of antibodies for human therapeutic use. In these applications, a truncated rSPA containing affinity chromatography resin is contacted with a solution containing a therapeutic antibody as well as undesired contaminating materials to selectively bind the desired antibody to the immobilized rSPA. The rSPA containing affinity chromatography resin with the desired antibody tightly bound to it is first washed to remove the contaminating materials, and then the antibody is eluted from the affinity chromatography resin in purified form by, for example, the use of acidic or high salt elution buffers.
[0078] The commercial significance of the therapeutic antibody market is expected to grow quickly in the near future. For example, the global market for therapeutic monoclonal antibodies in 2002 was reportedly above $5 billion and has been projected to approximately triple in size by 2008 to nearly $17 billion. Reichert and Pavlou, Nature Reviews Drug Discovery, 3:383-4, 2004. Servicing this market will be benefited by cost-effective tools for large scale, reliable purification of monoclonal antibodies.
[0079] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
EXAMPLES
Materials and Methods
[0080] All enzymes used in the procedures described herein were purchased from New England BioLabs (Ipswich, Mass.). All DNA purification kits were purchased from Qiagen, Inc (Valencia, Calif.). All agarose plates were purchased from Teknova, Inc (Hollister, Calif.). The engineered rSPA-s was synthesized and supplied in the plasmid pJ5:G03257. All E. coli hosts were lab strains with the exception of Subcloning Efficiency® DH5α cells, which were purchased from Invitrogen, Inc (Carlsbad, Calif.).
[0081] Preparation of Plasmid DNA Stocks
[0082] All liquid cultures were grown overnight for 12-16 hours at 37° C., shaking at 250 RPM in disposable plastic baffled-bottom flasks. All plasmid DNA was isolated using the QIAGEN Plasmid Maxi kit according to manufacturers directions.
[0083] The pREV2.1 vector described in WO 90/03984 was digested with HpaI and NruI endonucleases to release the β-glucuronidase signal sequence and a small 3' portion of the β-glucuronidase promoter. The vector was then re-ligated and subsequently digested with MluI and BamHI. As described below, the PCR amplified optimized protein A coding sequence was digested MluI and BamHI and cloned by ligation with T4 ligase into digested pREV2.1 vector to yield the construct pREV2.1-rSPA. FIG. 8 shows a (partial) DNA sequence of the construct, in which the vector sequence is indicated by italics, the promoter sequence is underlined, and the start methionine and the termination codons are in bold.
[0084] The pREV2.1-rSPA plasmid served as a source of the pREV2.1 plasmid backbone. A vial of PR13/pREV2.1-rSPA was thawed and used to inoculate 100 mL of Miller LB media (BD Biosciences, San Jose, Calif.) with 34 μg/mL chloramphenicol and 100 μg/mL ampicillin. A HMS174/pET12a glycerol stock was scratched and used to inoculate 100 mL of LB media containing 100 μg/mL of ampicillin.
[0085] Preparation of Transformation Competent E. Coli Host Cells
[0086] Both PR13 and BL21(DE3) host E. coli (Table 1) were prepared according to an adapted CaCl2 protocol (Elec. J. Biotech., 8:114-120). Briefly, cells were grown under selection to an OD600 ˜0.25-0.4, then harvested in 50 mL Oak Ridge tubes (Nalgene, Rochester, N.Y.). Pellets were resuspended in one-half volume ice-cold TB solution (10 mM PIPES, 75 mM CaCl2.2H2O, 250 mM KCl, 55 mM MnCl2.4H2O, pH 6.7 with KOH) and incubated on ice for 25 minutes. Cells were centrifuged at 8,000 RPM for 1 minute at 4° C., TB was decanted and pellet was resuspended in one-tenth original volume ice-cold TB. Aliquots of 100 μL were snap frozen in liquid nitrogen and stored at -80° C.
TABLE-US-00002 TABLE 1 E. coli strains described herein Strain Description PR13 Strain of E. coli used for protein expression with pREV2.1 expression system (F- thr-1 leuB6(Am) lacY1 rna-19 LAMpnp-13 rpsL132(StrR) malT1(LamR) xyl-7 mtlA2 thi-1) BLR(DE3) Strain of E. coli used with pET expression system (E. coli B F- ompT hsdSB(rB- mB-)gal dcm (DE3) Δ(srlrecA)306::Tn10 (TetR) DH5α Strain of E. coli used primarily for plasmid maintenance (F- φ80lacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rk-, mk+) phoA supE44 thi-1 gyrA96 relA1 λ-)
Example 1
Recombinant DNA Construct for Expression in E. Coli
[0087] To manufacture truncated rSPA in a host that is less pathogenic than S. aureus, a DNA construct was engineered to express a truncated version of strain 8325-4 protein A in E. coli. The construct contained a truncated 8325-4 protein A coding sequence including the E, D, A, B, C, and part of the X domains, but missing both the native N-terminal S. aureus signal sequence and a portion of the native C-terminal X domain. The DNA construct did not introduce coding sequences for heterologous polypeptides not found in native SPA. The coding sequence was functionally linked to an E. coli promoter and E. coli ribosome binding site. Restriction digestions and ligations were performed according to manufacturer instructions. PCR amplifications were performed as described below. Ligations were transformed into E. coli strain DH5α. DNA sequencing of one of a DH5α clone (18A) was performed under contract with the Iowa State University Sequencing Facility (Ames, Iowa) using the following primers: IPA-1: 5' AAA GCA GAT GCT CAG CAA (SEQ ID NO:18); IPA-2: 5' GAT TTC CTT GCT GAC GCT T (SEQ ID NO:19); Anti-IPA-2: 5' AAG CGT CAG CAA GGAAAT C (SEQ ID NO:20); and BG promoter-2: 5' GAT CTA TAT CAC GCT GTG G (SEQ ID NO:21).
Example 2
Expression of Truncated rSPA in E. Coli
[0088] The ability of four independent DH5α clones (labeled PA/pREV 7a, 9a, 10a, and 18a) harboring the construct described in Example 1 to express recombinant truncated rSPA was evaluated by SDS-PAGE and Western Blotting. Total cell lysates from E. coli were electrophoresed on SDS-PAGE gels. Samples were analyzed by SDS-PAGE as described above.
[0089] SDS-PAGE results were consistent with the predicted molecular mass of ˜47 kDA for recombinant truncated rSPA encoded by PA/pREV (FIG. 8). The results indicate that the constructs described herein can be abundantly expressed in E. coli without substantial degradation.
Example 3
Functional Characterization of Truncated rSPA Recovered from E. Coli
[0090] Truncated rSPA (SEQ ID NO: 7) was attached to a controlled glass pore resin (CPG-PA) and its functional characteristics were evaluated in a number of tests. To make the rSPA resin, truncated rSPA (from clone 18a in Example 2) was harvested and purified. Truncated rSPA was fused to control pore glass beads and functional characteristics were compared to those of Millipore's PROSEP® A High Capacity protein A controlled pore glass resin (Catalog No. 113115824).
[0091] Static Binding Assay
[0092] A static polyclonal binding assay was performed by equilibrating resin with phosphate buffered saline (PBS) buffer pH 7.2. Polyclonal human IgG (hIgG) was added and allowed to incubate at room temperature for 30 minutes with end over end mixing. The resin was washed with PBS buffer pH 7.2. The hIgG protein was eluted with 0.2 M Glycine pH 2.0. The amount of hIgG in the eluate was determined by measuring UV adsorbance at 280 nm, and the binding capacity calculated. The assay was performed three consecutive times, using the same glass-bound protein A samples to determine the persistent binding capacity of each product after repeated use.
[0093] Results of the static binding assay indicate that CPG-rSPA has a similar static binding capacity to that of the PROSEP® A product. Binding capacity was determined to be 36.9+0.2 mg IgG per ml of CPG-rSPA resin compared to 35.2+0.4 mg IgG per ml resin of PROSEP® A product in the first cycle. The results in Table 2 indicate that after three consecutive binding experiments, neither product suffered significant reduction of binding capacity.
TABLE-US-00003 TABLE 2 Result (mg IgG/ml resin) Sample Cycle 1 Cycle 2 Cycle 3 CPG-rSPA 36.9 ± 0.2 36.2 ± 0.4 36.0 ± 0.6 PROSEP A 35.2 ± 0.4 35.4 ± 1.2 34.7 ± 1.0
[0094] Protein A Leaching
[0095] A protein A ELISA kit (from Repligen) was used to quantify the amount of protein A that leached into the eluates used to determine the static binding capacity shown in Table 2. ELISAs were performed as indicated by the manufacturer.
[0096] Results in Table 3 indicate that less protein A leached into the first cycle eluate from CPG-rSPA than from the PROSEP® A product. In second and third cycles, protein A leaching was comparable for both protein A resins.
TABLE-US-00004 TABLE 3 Result (ng PA/mg hIgG) Sample Cycle 1 Cycle 2 Cycle 3 CPG-rSPA 14.1 ± 3.8 10 ± 1.1 11.4 ± 4.7 PROSEP A 31.9 ± 8.4 16.3 ± 2.1 10.1 ± 1.4
[0097] Capacity Following Cleaning and Regeneration Exposure Cycles
[0098] Binding capacity for CPG-rSPA and PROSEP A resins were evaluated subsequent to regeneration and cleaning Resins were washed with 0.3% HCl pH 1.5 and then exposed for 1 hour to 6 M Guanidine. Guanidine was removed by washing resins with 0.3% HCl pH 1.5, followed by an incubation period of 1 hour in the HCL solution. Following cleaning, each resin was equilibrated with PBS and the static hIgG binding capacity was measured as described above in section 1 (Static Binding Assay).
[0099] Results in Table 4 show no meaningful decrease in the binding capacity of the CPG-rSPA following three repeated cycles of HCL and Guanidine exposure consistent with PROSEP® A HC results.
TABLE-US-00005 TABLE 4 Result (mg IgG/ml resin) Sample Pre-clean Cycle 1 Cycle 2 Cycle 3 CPG-rSPA 35.2 ± 0.2 35.1 ± 0.2 32.9 ± 1.0 33.3 ± 0.8 PROSEP A 34.8 ± 1.2 31.9 ± 0.4 35.4 ± 2.52 34.6 ± 0.8
[0100] Non-Specific Protein Adsorption Following Cleaning and Regeneration
[0101] Each resin was incubated with Chinese Hamster Ovary (CHO) K1 cell conditioned medium containing 5% FBS at room temperature for 30 minutes. The resin was washed with PBS and then eluted with glycine pH 2.0 and neutralized with Tris buffer. Eluates were analyzed by (i) SDS-PAGE and silver staining the protein gels and (ii) a CHO host Protein ELISA (Cygnus Technologies).
[0102] SDS-PAGE showed several non-specific protein bands for both CPG-rSPA and the PROSEP® A HC that were similar in molecular weight and intensity (Data not shown). ELISA assay was not able to quantify bound host CHO proteins, indicating that both resins bind less than less than 5 ng CHO Protein/mg hIgG, the limit of detection for the assay.
[0103] Dynamic Binding Capacity
[0104] Dynamic binding breakthrough curves were generated by subjecting CPG-rSPA and PROSEP A to flow velocities of 100, 300, 500, and 700 cm/hr. A feed stream of 1.0 mg/ml polyclonal human IgG was used with a resin volume of 0.5 ml and a column bed height of 2.5 cm. Capacity is reported at 10% breakthrough.
[0105] Under the conditions tested, CPG-PA performed comparably to PROSEP® A HC at each flow velocity. See Table 5 and FIG. 10.
TABLE-US-00006 TABLE 5 Capacity at 10% BT (mg IgG/ml resin) Sample 100 cm/hr 300 cm/hr 500 cm/hr 700 cm/hr CPG-rSPA 19.5 6.2 6.4 5.3 PROSEP A 20 6.9 6.9 5.3
[0106] The results of the functional comparative analysis described herein indicate that recombinant truncated rSPA expressed in E. coli, when attached to a controlled pore glass resin, performed at least as well and, in some cases better, than Millipore's PROSEP A product, which incorporates an SPA ligand derived from native S. aureus.
Example 4
Functional Advantage of a Truncated X Domain
[0107] This example demonstrates the advantage of rSPA containing a truncated X domain compared to rSPA without an X domain on chromatography resin immunoglobulin binding capacities. rSPA and a Protein A (TPA), which contains the five Immunoglobulin binding domains, but does not contain any of the X domain, were immobilized onto SEPHAROSE® 4 Fast Flow resin (Amersham). The protein immobilizations were performed using equivalent molar concentrations of each protein A under identical conditions. A static polyclonal human IgG (hIgG) binding assay was performed as previously described. The rSPA immobilized product had a 14 to 22% greater hIgG capacity than the X domain deficient TPA (see Table 6).
TABLE-US-00007 TABLE 6 Protein A Concentration Static hIgG Binding Sample [uM] Capacity [g/L] TPA 50 15.5 + 0.5 rSPA 50 18.8 + 0.7 TPA 200 41.4 + 1.7 rSPA 200 47.2 + 1.1
Other Embodiments
[0108] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
Sequence CWU
1
2411272DNAArtificial SequenceSynthetically generated oligonucleotide
1atggcgcaac acgatgaagc tcaacagaac gctttttacc aggtactgaa catgccgaac
60ctgaacgcgg atcagcgcaa cggtttcatc cagagcctga aagacgaccc ttctcagtcc
120gcaaacgttc tgggcgaggc tcagaaactg aacgacagcc aggccccaaa agcagatgct
180cagcaaaata acttcaacaa ggaccagcag agcgcattct acgaaatcct gaacatgcca
240aatctgaacg aagctcaacg caacggcttc attcagtctc tgaaagacga tccgtcccag
300tccactaacg ttctgggtga agctaagaag ctgaacgaat cccaggcacc aaaagcagac
360aacaacttca acaaagagca gcagaacgct ttctatgaaa tcttgaacat gcctaacctg
420aatgaagaac agcgtaacgg cttcatccag tctctgaagg acgaccctag ccagtctgct
480aacctgctgt ccgaagcaaa aaaactgaac gagtcccagg ctccaaaagc ggataacaaa
540ttcaacaagg agcagcagaa cgcattctac gaaatcctgc acctgccgaa cctgaacgaa
600gaacagcgta acggtttcat ccaatccctg aaagacgatc cttcccagtc cgcaaatctg
660ctggcagaag caaagaaact gaacgacgca caggcaccga aggctgacaa caagttcaac
720aaagagcagc agaatgcctt ctacgagatt ctgcatctgc caaacctgac tgaggagcag
780cgcaacggtt tcattcagtc cctgaaggac gacccaagcg tcagcaagga aatcctggct
840gaggcgaaaa aactgaacga tgcacaggct ccgaaggaag aagacaacaa taaacctggt
900aaagaagata ataataagcc tggcaaggaa gataacaaca agccgggcaa ggaggacaac
960aataaaccgg gcaaagagga taataacaag cctggtaagg aagacaacaa caaaccaggc
1020aaagaagatg gcaacaagcc gggtaaggag gataataaaa aaccaggcaa ggaagacggc
1080aacaaacctg gcaaggagga taacaaaaag ccaggcaagg aggatggtaa taaaccgggc
1140aaagaagacg gcaacaagcc tggtaaagaa gacggtaacg gtgtacacgt cgttaaacct
1200ggtgacaccg tgaacgacat cgctaaggct aatggcacca cggcagacaa gattgcagcg
1260gacaataaat aa
127221523DNAArtificial SequenceSynthetically generated oligonucleotide
2atggcgcaac acgatgaagc tcaccagaac gctttttacc aggtactgaa catgccgaac
60ctgaacgcgg atcagcgcaa cggtttcatc cagagcctga aagacgaccc ttctcagtcc
120gcaaacgttc tgggcgaggc tcagaaactg aacgacagcc aggccccaaa agcagatgct
180cagcaaaata acttcaacaa ggaccagcag agcgcattct acgaaatcct gaacatgcca
240aatctgaacg aagctcaacg caacggcttc attcagtctc tgaaagacga tccgtcccag
300tccactaacg ttctgggtga agctaagaag ctgaacgaat cccaggcacc aaaagcagac
360aacaacttca acaaagagca gcagaacgct ttctatgaaa tcttgaacat gcctaacctg
420aatgaagaac agcgtaacgg cttcatccag tctctgaagg acgaccctag ccagtctgct
480aacctgctgt ccgaagcaaa aaaactgaac gagtcccagg ctccaaaagc ggataacaaa
540ttcaacaagg agcagcagaa cgcattctac gaaatcctgc acctgccgaa cctgaacgaa
600gaacagcgta acggtttcat ccaatccctg aaagacgatc cttcccagtc cgcaaatctg
660ctggcagaag caaagaaact gaacgacgca caggcaccga aggctgacaa caagttcaac
720aaagagcagc agaatgcctt ctacgagatt ctgcatctgc caaacctgac tgaggagcag
780cgcaacggtt tcattcagtc cctgaaggac gacccaagcg tcagcaagga aatcctggct
840gaggcgaaaa aactgaacga tgcacaggct ccgaaggaag aagacaacaa taaacctggt
900aaagaagata ataataagcc tggcaaggaa gataacaaca agccgggcaa ggaggacaac
960aataaaccgg gcaaagagga taataacaag cctggtaagg aagacaacaa caaaccaggc
1020aaagaagatg gcaacaagcc gggtaaggag gataataaaa aaccaggcaa ggaagacggc
1080aacaaacctg gcaaggagga taacaaaaag ccaggcaagg aggatggtaa taaaccgggc
1140aaagaagacg gcaacaagcc tggtaaagaa gacggtaacg gtgtacacgt cgttaaacct
1200ggtgacaccg tgaacgacat cgctaaggct aatggcacca cggcagacaa gattgcagcg
1260gacaataaat tagctgataa ataaggatcc ggatccgtcg acaagcttcc cgggagctcg
1320aattcttgaa gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
1380ataatggttt cttagacgtc ggtaccagcc cgcctaatga gcgggctttt ttttgacgtc
1440aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
1500gaggaagaca acaacaagcc tgg
1523319DNAArtificial SequenceA bacterial ribosome binding site
3acgcgtggag gatgattaa
194524PRTStaphylococcus aureus 4Met Lys Lys Lys Asn Ile Tyr Ser Ile Arg
Lys Leu Gly Val Gly Ile1 5 10
15Ala Ser Val Thr Leu Gly Thr Leu Leu Ile Ser Gly Gly Val Thr Pro
20 25 30Ala Ala Asn Ala Ala Gln
His Asp Glu Ala Gln Gln Asn Ala Phe Tyr 35 40
45Gln Val Leu Asn Met Pro Asn Leu Asn Ala Asp Gln Arg Asn
Gly Phe 50 55 60Ile Gln Ser Leu Lys
Asp Asp Pro Ser Gln Ser Ala Asn Val Leu Gly65 70
75 80Glu Ala Gln Lys Leu Asn Asp Ser Gln Ala
Pro Lys Ala Asp Ala Gln 85 90
95Gln Asn Asn Phe Asn Lys Asp Gln Gln Ser Ala Phe Tyr Glu Ile Leu
100 105 110Asn Met Pro Asn Leu
Asn Glu Ala Gln Arg Asn Gly Phe Ile Gln Ser 115
120 125Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu
Gly Glu Ala Lys 130 135 140Lys Leu Asn
Glu Ser Gln Ala Pro Lys Ala Asp Asn Asn Phe Asn Lys145
150 155 160Glu Gln Gln Asn Ala Phe Tyr
Glu Ile Leu Asn Met Pro Asn Leu Asn 165
170 175Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys
Asp Asp Pro Ser 180 185 190Gln
Ser Ala Asn Leu Leu Ser Glu Ala Lys Lys Leu Asn Glu Ser Gln 195
200 205Ala Pro Lys Ala Asp Asn Lys Phe Asn
Lys Glu Gln Gln Asn Ala Phe 210 215
220Tyr Glu Ile Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly225
230 235 240Phe Ile Gln Ser
Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu 245
250 255Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln
Ala Pro Lys Ala Asp Asn 260 265
270Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu
275 280 285Pro Asn Leu Thr Glu Glu Gln
Arg Asn Gly Phe Ile Gln Ser Leu Lys 290 295
300Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys
Leu305 310 315 320Asn Asp
Ala Gln Ala Pro Lys Glu Glu Asp Asn Asn Lys Pro Gly Lys
325 330 335Glu Asp Asn Asn Lys Pro Gly
Lys Glu Asp Asn Asn Lys Pro Gly Lys 340 345
350Glu Asp Asn Asn Lys Pro Gly Lys Glu Asp Asn Asn Lys Pro
Gly Lys 355 360 365Glu Asp Asn Asn
Lys Pro Gly Lys Glu Asp Gly Asn Lys Pro Gly Lys 370
375 380Glu Asp Asn Lys Lys Pro Gly Lys Glu Asp Gly Asn
Lys Pro Gly Lys385 390 395
400Glu Asp Asn Lys Lys Pro Gly Lys Glu Asp Gly Asn Lys Pro Gly Lys
405 410 415Glu Asp Gly Asn Lys
Pro Gly Lys Glu Asp Gly Asn Gly Val His Val 420
425 430Val Lys Pro Gly Asp Thr Val Asn Asp Ile Ala Lys
Ala Asn Gly Thr 435 440 445Thr Ala
Asp Lys Ile Ala Ala Asp Asn Lys Leu Ala Asp Lys Asn Met 450
455 460Ile Lys Pro Gly Gln Glu Leu Val Val Asp Lys
Lys Gln Pro Ala Asn465 470 475
480His Ala Asp Ala Asn Lys Ala Gln Ala Leu Pro Glu Thr Gly Glu Glu
485 490 495Asn Pro Phe Ile
Gly Thr Thr Val Phe Gly Gly Leu Ser Leu Ala Leu 500
505 510Gly Ala Ala Leu Leu Ala Gly Arg Arg Arg Glu
Leu 515 5205422PRTArtificial SequenceSynthetically
generated peptide 5Ala Gln His Asp Glu Ala Gln Gln Asn Ala Phe Tyr Gln
Val Leu Asn1 5 10 15Met
Pro Asn Leu Asn Ala Asp Gln Arg Asn Gly Phe Ile Gln Ser Leu 20
25 30Lys Asp Asp Pro Ser Gln Ser Ala
Asn Val Leu Gly Glu Ala Gln Lys 35 40
45Leu Asn Asp Ser Gln Ala Pro Lys Ala Asp Ala Gln Gln Asn Asn Phe
50 55 60Asn Lys Asp Gln Gln Ser Ala Phe
Tyr Glu Ile Leu Asn Met Pro Asn65 70 75
80Leu Asn Glu Ala Gln Arg Asn Gly Phe Ile Gln Ser Leu
Lys Asp Asp 85 90 95Pro
Ser Gln Ser Thr Asn Val Leu Gly Glu Ala Lys Lys Leu Asn Glu
100 105 110Ser Gln Ala Pro Lys Ala Asp
Asn Asn Phe Asn Lys Glu Gln Gln Asn 115 120
125Ala Phe Tyr Glu Ile Leu Asn Met Pro Asn Leu Asn Glu Glu Gln
Arg 130 135 140Asn Gly Phe Ile Gln Ser
Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn145 150
155 160Leu Leu Ser Glu Ala Lys Lys Leu Asn Glu Ser
Gln Ala Pro Lys Ala 165 170
175Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu
180 185 190His Leu Pro Asn Leu Asn
Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser 195 200
205Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu
Ala Lys 210 215 220Lys Leu Asn Asp Ala
Gln Ala Pro Lys Ala Asp Asn Lys Phe Asn Lys225 230
235 240Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu
His Leu Pro Asn Leu Thr 245 250
255Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser
260 265 270Val Ser Lys Glu Ile
Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln 275
280 285Ala Pro Lys Glu Glu Asp Asn Asn Lys Pro Gly Lys
Glu Asp Asn Asn 290 295 300Lys Pro Gly
Lys Glu Asp Asn Asn Lys Pro Gly Lys Glu Asp Asn Asn305
310 315 320Lys Pro Gly Lys Glu Asp Asn
Asn Lys Pro Gly Lys Glu Asp Asn Asn 325
330 335Lys Pro Gly Lys Glu Asp Gly Asn Lys Pro Gly Lys
Glu Asp Asn Lys 340 345 350Lys
Pro Gly Lys Glu Asp Gly Asn Lys Pro Gly Lys Glu Asp Asn Lys 355
360 365Lys Pro Gly Lys Glu Asp Gly Asn Lys
Pro Gly Lys Glu Asp Gly Asn 370 375
380Lys Pro Gly Lys Glu Asp Gly Asn Gly Val His Val Val Lys Pro Gly385
390 395 400Asp Thr Val Asn
Asp Ile Ala Lys Ala Asn Gly Thr Thr Ala Asp Lys 405
410 415Ile Ala Ala Asp Asn Lys
4206458PRTArtificial SequenceSynthetically generated peptide 6Met Lys Lys
Lys Asn Ile Tyr Ser Ile Arg Lys Leu Gly Val Gly Ile1 5
10 15Ala Ser Val Thr Leu Gly Thr Leu Leu
Ile Ser Gly Gly Val Thr Pro 20 25
30Ala Ala Thr Ala Ala Gln His Asp Glu Ala Gln Gln Asn Ala Phe Tyr
35 40 45Gln Val Leu Asn Met Pro Asn
Leu Asn Ala Asp Gln Arg Asn Gly Phe 50 55
60Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Val Leu Gly65
70 75 80Glu Ala Gln Lys
Leu Asn Asp Ser Gln Ala Pro Lys Ala Asp Ala Gln 85
90 95Gln Asn Asn Phe Asn Lys Asp Gln Gln Ser
Ala Phe Tyr Glu Ile Leu 100 105
110Asn Met Pro Asn Leu Asn Glu Ala Gln Arg Asn Gly Phe Ile Gln Ser
115 120 125Leu Lys Asp Asp Pro Ser Gln
Ser Thr Asn Val Leu Gly Glu Ala Lys 130 135
140Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala Asp Asn Asn Phe Asn
Lys145 150 155 160Glu Gln
Gln Asn Ala Phe Tyr Glu Ile Leu Asn Met Pro Asn Leu Asn
165 170 175Glu Glu Gln Arg Asn Gly Phe
Ile Gln Ser Leu Lys Asp Asp Pro Ser 180 185
190Gln Ser Ala Asn Leu Leu Ser Glu Ala Lys Lys Leu Asn Glu
Ser Gln 195 200 205Ala Pro Lys Ala
Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe 210
215 220Tyr Glu Ile Leu His Leu Pro Asn Leu Asn Glu Glu
Gln Arg Asn Gly225 230 235
240Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu
245 250 255Ala Glu Ala Lys Lys
Leu Asn Asp Ala Gln Ala Pro Lys Ala Asp Asn 260
265 270Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu
Ile Leu His Leu 275 280 285Pro Asn
Leu Thr Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys 290
295 300Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala
Glu Ala Lys Lys Leu305 310 315
320Asn Asp Ala Gln Ala Pro Lys Glu Glu Asp Asn Asn Lys Pro Gly Lys
325 330 335Glu Asp Asn Asn
Lys Pro Gly Lys Glu Asp Asn Asn Lys Pro Gly Lys 340
345 350Glu Asp Asn Asn Lys Pro Gly Lys Glu Asp Asn
Asn Lys Pro Gly Lys 355 360 365Glu
Asp Asn Asn Lys Pro Gly Lys Glu Asp Gly Asn Lys Pro Gly Lys 370
375 380Glu Asp Asn Lys Lys Pro Gly Lys Glu Asp
Gly Asn Lys Pro Gly Lys385 390 395
400Glu Asp Asn Lys Lys Pro Gly Lys Glu Asp Gly Asn Lys Pro Gly
Lys 405 410 415Glu Asp Gly
Asn Lys Pro Gly Lys Glu Asp Gly Asn Gly Val His Val 420
425 430Val Lys Pro Gly Asp Thr Val Asn Asp Ile
Ala Lys Ala Asn Gly Thr 435 440
445Thr Ala Asp Lys Ile Ala Ala Asp Asn Lys 450
4557426PRTArtificial SequenceSynthetically generated peptide 7Ala Gln His
Asp Glu Ala Gln Gln Asn Ala Phe Tyr Gln Val Leu Asn1 5
10 15Met Pro Asn Leu Asn Ala Asp Gln Arg
Asn Gly Phe Ile Gln Ser Leu 20 25
30Lys Asp Asp Pro Ser Gln Ser Ala Asn Val Leu Gly Glu Ala Gln Lys
35 40 45Leu Asn Asp Ser Gln Ala Pro
Lys Ala Asp Ala Gln Gln Asn Asn Phe 50 55
60Asn Lys Asp Gln Gln Ser Ala Phe Tyr Glu Ile Leu Asn Met Pro Asn65
70 75 80Leu Asn Glu Ala
Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp 85
90 95Pro Ser Gln Ser Thr Asn Val Leu Gly Glu
Ala Lys Lys Leu Asn Glu 100 105
110Ser Gln Ala Pro Lys Ala Asp Asn Asn Phe Asn Lys Glu Gln Gln Asn
115 120 125Ala Phe Tyr Glu Ile Leu Asn
Met Pro Asn Leu Asn Glu Glu Gln Arg 130 135
140Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala
Asn145 150 155 160Leu Leu
Ser Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala
165 170 175Asp Asn Lys Phe Asn Lys Glu
Gln Gln Asn Ala Phe Tyr Glu Ile Leu 180 185
190His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe Ile
Gln Ser 195 200 205Leu Lys Asp Asp
Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala Lys 210
215 220Lys Leu Asn Asp Ala Gln Ala Pro Lys Ala Asp Asn
Lys Phe Asn Lys225 230 235
240Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Thr
245 250 255Glu Glu Gln Arg Asn
Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser 260
265 270Val Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu
Asn Asp Ala Gln 275 280 285Ala Pro
Lys Glu Glu Asp Asn Asn Lys Pro Gly Lys Glu Asp Asn Asn 290
295 300Lys Pro Gly Lys Glu Asp Asn Asn Lys Pro Gly
Lys Glu Asp Asn Asn305 310 315
320Lys Pro Gly Lys Glu Asp Asn Asn Lys Pro Gly Lys Glu Asp Asn Asn
325 330 335Lys Pro Gly Lys
Glu Asp Gly Asn Lys Pro Gly Lys Glu Asp Asn Lys 340
345 350Lys Pro Gly Lys Glu Asp Gly Asn Lys Pro Gly
Lys Glu Asp Asn Lys 355 360 365Lys
Pro Gly Lys Glu Asp Gly Asn Lys Pro Gly Lys Glu Asp Gly Asn 370
375 380Lys Pro Gly Lys Glu Asp Gly Asn Gly Val
His Val Val Lys Pro Gly385 390 395
400Asp Thr Val Asn Asp Ile Ala Lys Ala Asn Gly Thr Thr Ala Asp
Lys 405 410 415Ile Ala Ala
Asp Asn Lys Leu Ala Asp Lys 420
42588PRTArtificial SequenceSynthetically generated peptide 8Lys Pro Gly
Lys Glu Asp Xaa Xaa1 5956PRTArtificial
SequenceSynthetically generated peptide 9Ala Gln His Asp Glu Ala Gln Gln
Asn Ala Phe Tyr Gln Val Leu Asn1 5 10
15Met Pro Asn Leu Asn Ala Asp Gln Arg Asn Gly Phe Ile Gln
Ser Leu 20 25 30Lys Asp Asp
Pro Ser Gln Ser Ala Asn Val Leu Gly Glu Ala Gln Lys 35
40 45Leu Asn Asp Ser Gln Ala Pro Lys 50
551061PRTArtificial SequenceSynthetically generated peptide 10Ala
Asp Ala Gln Gln Asn Asn Phe Asn Lys Asp Gln Gln Ser Ala Phe1
5 10 15Tyr Glu Ile Leu Asn Met Pro
Asn Leu Asn Glu Ala Gln Arg Asn Gly 20 25
30Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn
Val Leu 35 40 45Gly Glu Ala Lys
Lys Leu Asn Glu Ser Gln Ala Pro Lys 50 55
601158PRTArtificial SequenceSynthetically generated peptide 11Ala
Asp Asn Asn Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile1
5 10 15Leu Asn Met Pro Asn Leu Asn
Glu Glu Gln Arg Asn Gly Phe Ile Gln 20 25
30Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ser
Glu Ala 35 40 45Lys Lys Leu Asn
Glu Ser Gln Ala Pro Lys 50 551258PRTArtificial
SequenceSynthetically generated peptide 12Ala Asp Asn Lys Phe Asn Lys Glu
Gln Gln Asn Ala Phe Tyr Glu Ile1 5 10
15Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe
Ile Gln 20 25 30Ser Leu Lys
Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala 35
40 45Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys 50
551358PRTArtificial SequenceSynthetically generated
peptide 13Ala Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu
Ile1 5 10 15Leu His Leu
Pro Asn Leu Thr Glu Glu Gln Arg Asn Gly Phe Ile Gln 20
25 30Ser Leu Lys Asp Asp Pro Ser Val Ser Lys
Glu Ile Leu Ala Glu Ala 35 40
45Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys 50
5514180PRTArtificial SequenceSynthetically generated peptide 14Glu Glu
Asp Asn Asn Lys Pro Gly Lys Glu Asp Asn Asn Lys Pro Gly1 5
10 15Lys Glu Asp Asn Asn Lys Pro Gly
Lys Glu Asp Asn Asn Lys Pro Gly 20 25
30Lys Glu Asp Asn Asn Lys Pro Gly Lys Glu Asp Asn Asn Lys Pro
Gly 35 40 45Lys Glu Asp Gly Asn
Lys Pro Gly Lys Glu Asp Asn Lys Lys Pro Gly 50 55
60Lys Glu Asp Gly Asn Lys Pro Gly Lys Glu Asp Asn Lys Lys
Pro Gly65 70 75 80Lys
Glu Asp Gly Asn Lys Pro Gly Lys Glu Asp Gly Asn Lys Pro Gly
85 90 95Lys Glu Asp Gly Asn Gly Val
His Val Val Lys Pro Gly Asp Thr Val 100 105
110Asn Asp Ile Ala Lys Ala Asn Gly Thr Thr Ala Asp Lys Ile
Ala Ala 115 120 125Asp Asn Lys Leu
Ala Asp Lys Asn Met Ile Lys Pro Gly Gln Glu Leu 130
135 140Val Val Asp Lys Lys Gln Pro Ala Asn His Ala Asp
Ala Asn Lys Ala145 150 155
160Gln Ala Leu Pro Glu Thr Gly Glu Glu Asn Pro Phe Ile Gly Thr Thr
165 170 175Val Phe Gly Gly
18015131PRTArtificial SequenceSynthetically generated peptide 15Glu
Glu Asp Asn Asn Lys Pro Gly Lys Glu Asp Asn Asn Lys Pro Gly1
5 10 15Lys Glu Asp Asn Asn Lys Pro
Gly Lys Glu Asp Asn Asn Lys Pro Gly 20 25
30Lys Glu Asp Asn Asn Lys Pro Gly Lys Glu Asp Asn Asn Lys
Pro Gly 35 40 45Lys Glu Asp Gly
Asn Lys Pro Gly Lys Glu Asp Asn Lys Lys Pro Gly 50 55
60Lys Glu Asp Gly Asn Lys Pro Gly Lys Glu Asp Asn Lys
Lys Pro Gly65 70 75
80Lys Glu Asp Gly Asn Lys Pro Gly Lys Glu Asp Gly Asn Lys Pro Gly
85 90 95Lys Glu Asp Gly Asn Gly
Val His Val Val Lys Pro Gly Asp Thr Val 100
105 110Asn Asp Ile Ala Lys Ala Asn Gly Thr Thr Ala Asp
Lys Ile Ala Ala 115 120 125Asp Asn
Lys 130162270DNAArtificial SequenceA partial nucleotide sequence
of an E. coli expression vector. 16gcagcggtcg ggctgaacgg ggggttcgtg
cacacagccc agcttggagc gaacgaccta 60caccgaactg agatacctac agcgtgagct
atgagaaagc gccacgcttc ccgaagggag 120aaaggcggac aggtatccgg taagcggcag
ggtcggaaca ggagagcgca cgagggagct 180tccaggggga aacgcctggt atctttatag
tcctgtcggg tttcgccacc tctgacttga 240gcgtcgattt ttgtgatgct cgtcaggggg
gcggagccta tggaaaaacg ccagcaacgc 300ggccttttta cggttcctgg ccttttgctg
gccttttgct cacatgttct ttcctgcgtt 360atcccctgat tctgtggata accgtattac
cgcctttgag tgagctgata ccgctcgccg 420cagccgaacg accgagcgca gcgagtcagt
gagcgaggaa gcggaagagc gcctgatgcg 480gtattttctc cttacgcatc tgtgcggtat
ttcacaccgc atatgtcatg agagtttatc 540gttcccaata cgctcgaacg aacgttcggt
tgcttatttt atggcttctg tcaacgctgt 600tttaaagatt aatgcgatct atatcacgct
gtgggtattg cagtttttgg ttttttgatc 660gcggtgtcag ttctttttat ttccatttct
cttccatggg tttctcacag ataactgtgt 720gcaacacaga attggttcga acgcgtggag
gatgattaaa tggcgcaaca cgatgaagct 780caacagaacg ctttttacca ggtactgaac
atgccgaacc tgaacgcgga tcagcgcaac 840ggtttcatcc agagcctgaa agacgaccct
tctcagtccg caaacgttct gggcgaggct 900cagaaactga acgacagcca ggccccaaaa
gcagatgctc agcaaaataa cttcaacaag 960gaccagcaga gcgcattcta cgaaatcctg
aacatgccaa atctgaacga agctcaacgc 1020aacggcttca ttcagtctct gaaagacgat
ccgtcccagt ccactaacgt tctgggtgaa 1080gctaagaagc tgaacgaatc ccaggcacca
aaagcagaca acaacttcaa caaagagcag 1140cagaacgctt tctatgaaat cttgaacatg
cctaacctga atgaagaaca gcgtaacggc 1200ttcatccagt ctctgaagga cgaccctagc
cagtctgcta acctgctgtc cgaagcaaaa 1260aaactgaacg agtcccaggc tccaaaagcg
gataacaaat tcaacaagga gcagcagaac 1320gcattctacg aaatcctgca cctgccgaac
ctgaacgaag aacagcgtaa cggtttcatc 1380caatccctga aagacgatcc ttcccagtcc
gcaaatctgc tggcagaagc aaagaaactg 1440aacgacgcac aggcaccgaa ggctgacaac
aagttcaaca aagagcagca gaatgccttc 1500tacgagattc tgcatctgcc aaacctgact
gaggagcagc gcaacggttt cattcagtcc 1560ctgaaggacg acccaagcgt cagcaaggaa
atcctggctg aggcgaaaaa actgaacgat 1620gcacaggctc cgaaggaaga agacaacaat
aaacctggta aagaagataa taataagcct 1680ggcaaggaag ataacaacaa gccgggcaag
gaggacaaca ataaaccggg caaagaggat 1740aataacaagc ctggtaagga agacaacaac
aaaccaggca aagaagatgg caacaagccg 1800ggtaaggagg ataataaaaa accaggcaag
gaagacggca acaaacctgg caaggaggat 1860aacaaaaagc caggcaagga ggatggtaat
aaaccgggca aagaagacgg caacaagcct 1920ggtaaagaag acggtaacgg tgtacacgtc
gttaaacctg gtgacaccgt gaacgacatc 1980gctaaggcta atggcaccac ggcagacaag
attgcagcgg acaataaata aggatccgga 2040tccgtcgaca agcttcccgg gagctcgaat
tcttgaagac gaaagggcct cgtgatacgc 2100ctatttttat aggttaatgt catgataata
atggtttctt agacgtcggt accagcccgc 2160ctaatgagcg ggcttttttt tgacgtcagg
tggcactttt cggggaaatg tgcgcggaac 2220ccctatttgt ttatttttct aaatacagag
gaagacaaca acaagcctgg 227017471PRTArtificial
SequenceSynthetically generated peptide 17Ala Gln His Asp Glu Ala Gln Gln
Asn Ala Phe Tyr Gln Val Leu Asn1 5 10
15Met Pro Asn Leu Asn Ala Asp Gln Arg Asn Gly Phe Ile Gln
Ser Leu 20 25 30Lys Asp Asp
Pro Ser Gln Ser Ala Asn Val Leu Gly Glu Ala Gln Lys 35
40 45Leu Asn Asp Ser Gln Ala Pro Lys Ala Asp Ala
Gln Gln Asn Asn Phe 50 55 60Asn Lys
Asp Gln Gln Ser Ala Phe Tyr Glu Ile Leu Asn Met Pro Asn65
70 75 80Leu Asn Glu Ala Gln Arg Asn
Gly Phe Ile Gln Ser Leu Lys Asp Asp 85 90
95Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala Lys Lys
Leu Asn Glu 100 105 110Ser Gln
Ala Pro Lys Ala Asp Asn Asn Phe Asn Lys Glu Gln Gln Asn 115
120 125Ala Phe Tyr Glu Ile Leu Asn Met Pro Asn
Leu Asn Glu Glu Gln Arg 130 135 140Asn
Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn145
150 155 160Leu Leu Ser Glu Ala Lys
Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala 165
170 175Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe
Tyr Glu Ile Leu 180 185 190His
Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser 195
200 205Leu Lys Asp Asp Pro Ser Gln Ser Ala
Asn Leu Leu Ala Glu Ala Lys 210 215
220Lys Leu Asn Asp Ala Gln Ala Pro Lys Ala Asp Asn Lys Phe Asn Lys225
230 235 240Glu Gln Gln Asn
Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Thr 245
250 255Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser
Leu Lys Asp Asp Pro Ser 260 265
270Val Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln
275 280 285Ala Pro Lys Glu Glu Asp Asn
Asn Lys Pro Gly Lys Glu Asp Asn Asn 290 295
300Lys Pro Gly Lys Glu Asp Asn Asn Lys Pro Gly Lys Glu Asp Asn
Asn305 310 315 320Lys Pro
Gly Lys Glu Asp Asn Asn Lys Pro Gly Lys Glu Asp Asn Asn
325 330 335Lys Pro Gly Lys Glu Asp Gly
Asn Lys Pro Gly Lys Glu Asp Asn Lys 340 345
350Lys Pro Gly Lys Glu Asp Gly Asn Lys Pro Gly Lys Glu Asp
Asn Lys 355 360 365Lys Pro Gly Lys
Glu Asp Gly Asn Lys Pro Gly Lys Glu Asp Gly Asn 370
375 380Lys Pro Gly Lys Glu Asp Gly Asn Gly Val His Val
Val Lys Pro Gly385 390 395
400Asp Thr Val Asn Asp Ile Ala Lys Ala Asn Gly Thr Thr Ala Asp Lys
405 410 415Ile Ala Ala Asp Asn
Lys Leu Ala Asp Lys Asn Met Ile Lys Pro Gly 420
425 430Gln Glu Leu Val Val Asp Lys Lys Gln Pro Ala Asn
His Ala Asp Ala 435 440 445Asn Lys
Ala Gln Ala Leu Pro Glu Thr Gly Glu Glu Asn Pro Phe Ile 450
455 460Gly Thr Thr Val Phe Gly Gly465
4701818DNAArtificial SequencePrimer 18aaagcagatg ctcagcaa
181919DNAArtificial SequencePrimer
19gatttccttg ctgacgctt
192019DNAArtificial SequencePrimer 20aagcgtcagc aaggaaatc
192119DNAArtificial SequencePrimer
21gatctatatc acgctgtgg
19221269DNAArtificial SequenceSynthetically generated oligonucleotide
22atggcgcaac acgatgaagc tcaacaaaat gctttttatc aagtcttaaa tatgcctaac
60ttaaatgctg atcaacgcaa tggttttatc caaagcctta aagatgatcc aagccaaagt
120gctaacgttt taggtgaagc tcaaaaactt aatgactctc aagctccaaa agctgatgcg
180caacaaaata acttcaacaa agatcaacaa agcgccttct atgaaatctt gaacatgcct
240aacttaaacg aagcgcaacg taacggcttc attcaaagtc ttaaagacga cccaagccaa
300agcactaacg ttttaggtga agctaaaaaa ttaaacgaat ctcaagcacc gaaagctgat
360aacaatttca acaaagaaca acaaaatgct ttctatgaaa tcttgaatat gcctaactta
420aacgaagaac aacgcaatgg tttcatccaa agcttaaaag atgacccaag ccaaagtgct
480aacctattgt cagaagctaa aaagttaaat gaatctcaag caccgaaagc ggataacaaa
540ttcaacaaag aacaacaaaa tgctttctat gaaatcttac atttacctaa cttaaacgaa
600gaacaacgca atggtttcat ccaaagccta aaagatgacc caagccaaag cgctaacctt
660ttagcagaag ctaaaaagct aaatgatgct caagcaccaa aagctgacaa caaattcaac
720aaagaacaac aaaatgcttt ctatgaaatt ttacatttac ctaacttaac tgaagaacaa
780cgtaacggct tcatccaaag ccttaaagac gatccttcgg tgagcaaaga aattttagca
840gaagctaaaa agctaaacga tgctcaagca ccaaaagagg aagacaataa caagcctggc
900aaagaagaca ataacaagcc tggcaaagaa gacaataaca agcctggcaa agaagacaac
960aacaagcctg gcaaagaaga caacaacaag cctggtaaag aagacaacaa caagcctggc
1020aaagaagacg gcaacaagcc tggtaaagaa gacaacaaaa aacctggtaa agaagatggc
1080aacaagcctg gtaaagaaga caacaaaaaa cctggtaaag aagacggcaa caagcctggc
1140aaagaagatg gcaacaaacc tggtaaagaa gatggtaacg gagtacatgt cgttaaacct
1200ggtgatacag taaatgacat tgcaaaagca aacggcacta ctgctgacaa aattgctgca
1260gataacaaa
126923400DNAArtificial SequenceSynthetically generated oligonucleotide
23tctgtgcggt atttcacacc gcatatgtca tgagagttta tcgttcccaa tacgctcgaa
60cgaacgttcg gttgcttatt ttatggcttc tgtcaacgct gttttaaaga ttaatgcgat
120ctatatcacg ctgtgggtat tgcagttttt cgttttttga tcgcggtgtc agttcttttt
180atttccattt ctcttccatg ggtttctcac agataactgt gtgcaacaca gaattggttc
240gaacgcgtgg aggatgatta aatggcgcaa cacgatgaag ctcaacagaa cgctttttac
300caggtactga acatgccgaa ggacaataaa taaggatccg tcgacaagct tcccgggagc
360tcgaattctt gaagacgaaa gggcctcgtg atacgcctat
4002450PRTArtificial SequenceSynthetically generated peptide 24Glu Arg
Val Glu Asp Asp Met Ala Gln His Asp Glu Ala Gln Gln Asn1 5
10 15Ala Phe Tyr Gln Val Leu Asn Met
Pro Asn Asp Asn Lys Gly Ser Val 20 25
30Asp Lys Leu Pro Gly Ser Ser Asn Ser Arg Arg Lys Gly Leu Val
Ile 35 40 45Arg Leu 50
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