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. 1990 Apr 1;267(1):171–177. doi: 10.1042/bj2670171

Expression and purification of a truncated recombinant streptococcal protein G.

C R Goward 1, J P Murphy 1, T Atkinson 1, D A Barstow 1
PMCID: PMC1131260  PMID: 2183792

Abstract

The gene for Protein G from Streptococcus strain G148 was cloned and expressed in Escherichia coli. The regions on the gene corresponding to the albumin-binding domains and the Fab-binding region were then deleted by site-directed mutagenesis. The translation of regions corresponding to the cell-wall- and membrane-binding domains was prevented by introduction of stop codons upstream of these domains. This recombinant DNA sequence codes for a protein (G') that contains repetitive regions and that binds only the Fc portion of IgG, analogously to Protein A. Translation of the sequence produces a protein with an Mr of about 20,000. The nucleotide sequence differs from those published previously [Guss, Eliasson, Olsson, Uhlén, Frej, Jornvall, Flock & Lindberg (1986) EMBO J. 5, 1567-1575; Olsson, Eliasson, Guss, Nilsson, Hellman, Lindberg & Uhlén (1987) Eur. J. Biochem. 168, 319-324]. The protein can be substantially purified on a large scale by chromatography on IgG-Sepharose 4B. Homogeneous Protein G' can be prepared by anion-exchange f.p.l.c. on Mono Q HR. This Protein G' has a pI of 4.19 and SDS/PAGE gives an apparent anomalous Mr of 35,000.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Akerström B., Björck L. A physicochemical study of protein G, a molecule with unique immunoglobulin G-binding properties. J Biol Chem. 1986 Aug 5;261(22):10240–10247. [PubMed] [Google Scholar]
  2. Akerström B., Brodin T., Reis K., Björck L. Protein G: a powerful tool for binding and detection of monoclonal and polyclonal antibodies. J Immunol. 1985 Oct;135(4):2589–2592. [PubMed] [Google Scholar]
  3. Akerström B., Nielsen E., Björck L. Definition of IgG- and albumin-binding regions of streptococcal protein G. J Biol Chem. 1987 Oct 5;262(28):13388–13391. [PubMed] [Google Scholar]
  4. Björck L., Kastern W., Lindahl G., Widebäck K. Streptococcal protein G, expressed by streptococci or by Escherichia coli, has separate binding sites for human albumin and IgG. Mol Immunol. 1987 Oct;24(10):1113–1122. doi: 10.1016/0161-5890(87)90080-0. [DOI] [PubMed] [Google Scholar]
  5. Björck L., Kronvall G. Purification and some properties of streptococcal protein G, a novel IgG-binding reagent. J Immunol. 1984 Aug;133(2):969–974. [PubMed] [Google Scholar]
  6. Carter P., Bedouelle H., Winter G. Improved oligonucleotide site-directed mutagenesis using M13 vectors. Nucleic Acids Res. 1985 Jun 25;13(12):4431–4443. doi: 10.1093/nar/13.12.4431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Clewell D. B., Helinski D. R. Supercoiled circular DNA-protein complex in Escherichia coli: purification and induced conversion to an opern circular DNA form. Proc Natl Acad Sci U S A. 1969 Apr;62(4):1159–1166. doi: 10.1073/pnas.62.4.1159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cohen S. N., Chang A. C., Hsu L. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci U S A. 1972 Aug;69(8):2110–2114. doi: 10.1073/pnas.69.8.2110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Deininger P. L. Random subcloning of sonicated DNA: application to shotgun DNA sequence analysis. Anal Biochem. 1983 Feb 15;129(1):216–223. doi: 10.1016/0003-2697(83)90072-6. [DOI] [PubMed] [Google Scholar]
  10. Eliasson M., Andersson R., Olsson A., Wigzell H., Uhlén M. Differential IgG-binding characteristics of staphylococcal protein A, streptococcal protein G, and a chimeric protein AG. J Immunol. 1989 Jan 15;142(2):575–581. [PubMed] [Google Scholar]
  11. Eliasson M., Olsson A., Palmcrantz E., Wiberg K., Inganäs M., Guss B., Lindberg M., Uhlén M. Chimeric IgG-binding receptors engineered from staphylococcal protein A and streptococcal protein G. J Biol Chem. 1988 Mar 25;263(9):4323–4327. [PubMed] [Google Scholar]
  12. Erntell M., Myhre E. B., Sjöbring U., Björck L. Streptococcal protein G has affinity for both Fab- and Fc-fragments of human IgG. Mol Immunol. 1988 Feb;25(2):121–126. doi: 10.1016/0161-5890(88)90059-4. [DOI] [PubMed] [Google Scholar]
  13. Fahnestock S. R., Alexander P., Nagle J., Filpula D. Gene for an immunoglobulin-binding protein from a group G streptococcus. J Bacteriol. 1986 Sep;167(3):870–880. doi: 10.1128/jb.167.3.870-880.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Falkenberg C., Björck L., Akerström B., Nilsson S. Purification of streptococcal protein G expressed by Escherichia coli by high performance liquid affinity chromatography using immobilized immunoglobulin G and albumin. Biomed Chromatogr. 1987;2(5):221–225. doi: 10.1002/bmc.1130020510. [DOI] [PubMed] [Google Scholar]
  15. Filpula D., Alexander P., Fahnestock S. R. Nucleotide sequence of the protein G gene from Streptococcus GX7805, and comparison to previously reported sequences. Nucleic Acids Res. 1987 Sep 11;15(17):7210–7210. doi: 10.1093/nar/15.17.7210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Goding J. W. Use of staphylococcal protein A as an immunological reagent. J Immunol Methods. 1978;20:241–253. doi: 10.1016/0022-1759(78)90259-4. [DOI] [PubMed] [Google Scholar]
  17. Goward C. R., Barstow D. A. Solubilization of IgG-binding proteins from group A and G streptococci. Microbiol Immunol. 1989;33(2):123–127. doi: 10.1111/j.1348-0421.1989.tb01504.x. [DOI] [PubMed] [Google Scholar]
  18. Grunstein M., Hogness D. S. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3961–3965. doi: 10.1073/pnas.72.10.3961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Guss B., Eliasson M., Olsson A., Uhlén M., Frej A. K., Jörnvall H., Flock J. I., Lindberg M. Structure of the IgG-binding regions of streptococcal protein G. EMBO J. 1986 Jul;5(7):1567–1575. doi: 10.1002/j.1460-2075.1986.tb04398.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Guss B., Leander K., Hellman U., Uhlén M., Sjöquist J., Lindberg M. Analysis of protein A encoded by a mutated gene of Staphylococcus aureus Cowan I. Eur J Biochem. 1985 Dec 16;153(3):579–585. doi: 10.1111/j.1432-1033.1985.tb09340.x. [DOI] [PubMed] [Google Scholar]
  21. Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
  22. Hunkapiller M. W., Hewick R. M., Dreyer W. J., Hood L. E. High-sensitivity sequencing with a gas-phase sequenator. Methods Enzymol. 1983;91:399–413. doi: 10.1016/s0076-6879(83)91038-8. [DOI] [PubMed] [Google Scholar]
  23. Hunkapiller M. W., Hood L. E. Analysis of phenylthiohydantoins by ultrasensitive gradient high-performance liquid chromatography. Methods Enzymol. 1983;91:486–493. doi: 10.1016/s0076-6879(83)91045-5. [DOI] [PubMed] [Google Scholar]
  24. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  25. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  26. Langone J. J. Protein A of Staphylococcus aureus and related immunoglobulin receptors produced by streptococci and pneumonococci. Adv Immunol. 1982;32:157–252. [PubMed] [Google Scholar]
  27. McDonell M. W., Simon M. N., Studier F. W. Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J Mol Biol. 1977 Feb 15;110(1):119–146. doi: 10.1016/s0022-2836(77)80102-2. [DOI] [PubMed] [Google Scholar]
  28. Nygren P. A., Eliasson M., Abrahmsén L., Uhlén M., Palmcrantz E. Analysis and use of the serum albumin binding domains of streptococcal protein G. J Mol Recognit. 1988 Apr;1(2):69–74. doi: 10.1002/jmr.300010204. [DOI] [PubMed] [Google Scholar]
  29. Olsson A., Eliasson M., Guss B., Nilsson B., Hellman U., Lindberg M., Uhlén M. Structure and evolution of the repetitive gene encoding streptococcal protein G. Eur J Biochem. 1987 Oct 15;168(2):319–324. doi: 10.1111/j.1432-1033.1987.tb13423.x. [DOI] [PubMed] [Google Scholar]
  30. Radloff R., Bauer W., Vinograd J. A dye-buoyant-density method for the detection and isolation of closed circular duplex DNA: the closed circular DNA in HeLa cells. Proc Natl Acad Sci U S A. 1967 May;57(5):1514–1521. doi: 10.1073/pnas.57.5.1514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Reis K. J., Hansen H. F., Björck L. Extraction and characterization of IgG Fc receptors from group C and group G streptococci. Mol Immunol. 1986 Apr;23(4):425–431. doi: 10.1016/0161-5890(86)90140-9. [DOI] [PubMed] [Google Scholar]
  32. Sanger F., Coulson A. R., Barrell B. G., Smith A. J., Roe B. A. Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J Mol Biol. 1980 Oct 25;143(2):161–178. doi: 10.1016/0022-2836(80)90196-5. [DOI] [PubMed] [Google Scholar]
  33. Shuttleworth H. L., Duggleby C. J., Jones S. A., Atkinson T., Minton N. P. Nucleotide sequence analysis of the gene for protein A from Staphylococcus aureus Cowan 1 (NCTC8530) and its enhanced expression in Escherichia coli. Gene. 1987;58(2-3):283–295. doi: 10.1016/0378-1119(87)90383-0. [DOI] [PubMed] [Google Scholar]
  34. Sjodahl J. Repetitive sequences in protein A from Staphylococcus aureus. Arrangement of five regions within the protein, four being highly homologous and Fc-binding. Eur J Biochem. 1977 Mar 1;73(2):343–351. doi: 10.1111/j.1432-1033.1977.tb11324.x. [DOI] [PubMed] [Google Scholar]
  35. Sjöbring U., Björck L., Kastern W. Protein G genes: structure and distribution of IgG-binding and albumin-binding domains. Mol Microbiol. 1989 Mar;3(3):319–327. doi: 10.1111/j.1365-2958.1989.tb00177.x. [DOI] [PubMed] [Google Scholar]
  36. Sjöbring U., Falkenberg C., Nielsen E., Akerström B., Björck L. Isolation and characterization of a 14-kDa albumin-binding fragment of streptococcal protein G. J Immunol. 1988 Mar 1;140(5):1595–1599. [PubMed] [Google Scholar]
  37. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  38. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Uhlén M., Guss B., Nilsson B., Gatenbeck S., Philipson L., Lindberg M. Complete sequence of the staphylococcal gene encoding protein A. A gene evolved through multiple duplications. J Biol Chem. 1984 Feb 10;259(3):1695–1702. [PubMed] [Google Scholar]
  40. Uhlén M., Nilsson B., Guss B., Lindberg M., Gatenbeck S., Philipson L. Gene fusion vectors based on the gene for staphylococcal protein A. Gene. 1983 Sep;23(3):369–378. doi: 10.1016/0378-1119(83)90025-2. [DOI] [PubMed] [Google Scholar]
  41. Warnes A., Walkland A., Stephenson J. R. Development of an enzyme-linked immunosorbent assay for staphylococcal protein A produced in Escherichia coli by pUC8 based plasmids containing the Staphylococcus aureus Cowan I protein A gene. J Immunol Methods. 1986 Oct 23;93(1):63–70. doi: 10.1016/0022-1759(86)90433-3. [DOI] [PubMed] [Google Scholar]

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