Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1986 Sep;5(9):2393–2398. doi: 10.1002/j.1460-2075.1986.tb04509.x

Production of specific antibodies against protein A fusion proteins.

B Löwenadler, B Nilsson, L Abrahmsén, T Moks, L Ljungqvist, E Holmgren, S Paleus, S Josephson, L Philipson, M Uhlén
PMCID: PMC1167125  PMID: 3096719

Abstract

The gene for Staphylococcal protein A was fused to the coding sequence of bacterial beta-galactosidase, alkaline phosphatase and human insulin-like growth factor I (IGF-I). The fusion proteins, expressed in bacteria, were purified by affinity chromatography on IgG-Sepharose and antibodies were raised in rabbits. All three fusion proteins elicited specific antibodies against both the inserted protein sequences and the protein A moiety. In the case of IGF-I, the protein A moiety in the fusion protein may act as an adjuvant since native IGF-I alone is a poor immunogen. The results suggest that the protein A fusion system can be used for efficient antibody production against peptides or proteins expressed from cloned or synthetic genes. To facilitate such gene fusions a set of optimized vectors have been constructed.

Full text

PDF
2393

Images in this article

2394Fig. 2.
on p.2394
2395Fig. 3.
on p.2395
2395Fig. 4.
on p.2395
2396Fig. 6.
on p.2396

Selected References

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

  1. Arnon R., Maron E., Sela M., Anfinsen C. B. Antibodies reactive with native lysozyme elicited by a completely synthetic antigen. Proc Natl Acad Sci U S A. 1971 Jul;68(7):1450–1455. doi: 10.1073/pnas.68.7.1450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baxter R. C., Axiak S., Raison R. L. Monoclonal antibody against human somatomedin-C/insulin-like growth factor-I. J Clin Endocrinol Metab. 1982 Feb;54(2):474–476. doi: 10.1210/jcem-54-2-474. [DOI] [PubMed] [Google Scholar]
  3. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  4. Elmblad A., Josephson S., Palm G. Synthesis of mixed oligodeoxyribonucleotides following the solid phase method. Nucleic Acids Res. 1982 May 25;10(10):3291–3301. doi: 10.1093/nar/10.10.3291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Furlanetto R. W., Underwood L. E., Van Wyk J. J., D'Ercole A. J. Estimation of somatomedin-C levels in normals and patients with pituitary disease by radioimmunoassay. J Clin Invest. 1977 Sep;60(3):648–657. doi: 10.1172/JCI108816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Glass W. F., 2nd, Briggs R. C., Hnilica L. S. Identification of tissue-specific nuclear antigens transferred to nitrocellulose from polyacrylamide gels. Science. 1981 Jan 2;211(4477):70–72. doi: 10.1126/science.7003713. [DOI] [PubMed] [Google Scholar]
  7. Guss B., Uhlén M., Nilsson B., Lindberg M., Sjöquist J., Sjödahl J. Region X, the cell-wall-attachment part of staphylococcal protein A. Eur J Biochem. 1984 Jan 16;138(2):413–420. doi: 10.1111/j.1432-1033.1984.tb07931.x. [DOI] [PubMed] [Google Scholar]
  8. Götz F., Ahrné S., Lindberg M. Plasmid transfer and genetic recombination by protoplast fusion in staphylococci. J Bacteriol. 1981 Jan;145(1):74–81. doi: 10.1128/jb.145.1.74-81.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hoffman C. S., Wright A. Fusions of secreted proteins to alkaline phosphatase: an approach for studying protein secretion. Proc Natl Acad Sci U S A. 1985 Aug;82(15):5107–5111. doi: 10.1073/pnas.82.15.5107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hopp T. P., Woods K. R. Prediction of protein antigenic determinants from amino acid sequences. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3824–3828. doi: 10.1073/pnas.78.6.3824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Iordănescu S. Recombinant plasmid obtained from two different, compatible staphylococcal plasmids. J Bacteriol. 1975 Nov;124(2):597–601. doi: 10.1128/jb.124.2.597-601.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kasahara T., Kin K., Itoh Y., Kawai T., Morita M., Shioiri-Nakano K. Cellular cooperation in lymphocyte activation. IV. Requirement of cell-to-cell interaction for the activation of human T and B lymphocytes by protein A. Cell Immunol. 1980 Jan;49(1):142–153. doi: 10.1016/0008-8749(80)90064-7. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Laubli U. K., Baier W., Binz H., Celio M. R., Humbel R. E. Monoclonal antibodies directed to human insulin-like growth factor I (IGF I). Use for radioimmunoassay and immunopurification of IGF. FEBS Lett. 1982 Nov 22;149(1):109–112. doi: 10.1016/0014-5793(82)81082-x. [DOI] [PubMed] [Google Scholar]
  15. Lerner R. A., Green N., Alexander H., Liu F. T., Sutcliffe J. G., Shinnick T. M. Chemically synthesized peptides predicted from the nucleotide sequence of the hepatitis B virus genome elicit antibodies reactive with the native envelope protein of Dane particles. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3403–3407. doi: 10.1073/pnas.78.6.3403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lindmark R., Thorén-Tolling K., Sjöquist J. Binding of immunoglobulins to protein A and immunoglobulin levels in mammalian sera. J Immunol Methods. 1983 Aug 12;62(1):1–13. doi: 10.1016/0022-1759(83)90104-7. [DOI] [PubMed] [Google Scholar]
  17. Moks T., Abrahmsén L., Nilsson B., Hellman U., Sjöquist J., Uhlén M. Staphylococcal protein A consists of five IgG-binding domains. Eur J Biochem. 1986 May 2;156(3):637–643. doi: 10.1111/j.1432-1033.1986.tb09625.x. [DOI] [PubMed] [Google Scholar]
  18. Morrison D. A. Transformation and preservation of competent bacterial cells by freezing. Methods Enzymol. 1979;68:326–331. doi: 10.1016/0076-6879(79)68023-0. [DOI] [PubMed] [Google Scholar]
  19. Müller G. M., Shapira M., Arnon R. Anti-influenza response achieved by immunization with a synthetic conjugate. Proc Natl Acad Sci U S A. 1982 Jan;79(2):569–573. doi: 10.1073/pnas.79.2.569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Nilsson B., Abrahmsén L., Uhlén M. Immobilization and purification of enzymes with staphylococcal protein A gene fusion vectors. EMBO J. 1985 Apr;4(4):1075–1080. doi: 10.1002/j.1460-2075.1985.tb03741.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nilsson B., Holmgren E., Josephson S., Gatenbeck S., Philipson L., Uhlen M. Efficient secretion and purification of human insulin-like growth factor I with a gene fusion vector in Staphylococci. Nucleic Acids Res. 1985 Feb 25;13(4):1151–1162. doi: 10.1093/nar/13.4.1151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Nossal N. G., Heppel L. A. The release of enzymes by osmotic shock from Escherichia coli in exponential phase. J Biol Chem. 1966 Jul 10;241(13):3055–3062. [PubMed] [Google Scholar]
  23. Perkus M. E., Piccini A., Lipinskas B. R., Paoletti E. Recombinant vaccinia virus: immunization against multiple pathogens. Science. 1985 Sep 6;229(4717):981–984. doi: 10.1126/science.2992092. [DOI] [PubMed] [Google Scholar]
  24. Reed S. I. Preparation of product-specific antisera by gene fusion: antibodies specific for the product of the yeast cell-division-cycle gene CDC28. Gene. 1982 Dec;20(2):255–265. doi: 10.1016/0378-1119(82)90044-0. [DOI] [PubMed] [Google Scholar]
  25. Rinderknecht E., Humbel R. E. The amino acid sequence of human insulin-like growth factor I and its structural homology with proinsulin. J Biol Chem. 1978 Apr 25;253(8):2769–2776. [PubMed] [Google Scholar]
  26. Sjödahl J., Møller G. The Fc binding regions in protein A are not responsible for the polyclonal B cell activating property of protein A. Scand J Immunol. 1979;10(6):593–596. doi: 10.1111/j.1365-3083.1979.tb01394.x. [DOI] [PubMed] [Google Scholar]
  27. Sutcliffe J. G., Shinnick T. M., Green N., Liu F. T., Niman H. L., Lerner R. A. Chemical synthesis of a polypeptide predicted from nucleotide sequence allows detection of a new retroviral gene product. Nature. 1980 Oct 30;287(5785):801–805. doi: 10.1038/287801a0. [DOI] [PubMed] [Google Scholar]
  28. Tainer J. A., Getzoff E. D., Alexander H., Houghten R. A., Olson A. J., Lerner R. A., Hendrickson W. A. The reactivity of anti-peptide antibodies is a function of the atomic mobility of sites in a protein. Nature. 1984 Nov 8;312(5990):127–134. doi: 10.1038/312127a0. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. 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]
  32. Ullmann A. One-step purification of hybrid proteins which have beta-galactosidase activity. Gene. 1984 Jul-Aug;29(1-2):27–31. doi: 10.1016/0378-1119(84)90162-8. [DOI] [PubMed] [Google Scholar]
  33. Zabeau M., Stanley K. K. Enhanced expression of cro-beta-galactosidase fusion proteins under the control of the PR promoter of bacteriophage lambda. EMBO J. 1982;1(10):1217–1224. doi: 10.1002/j.1460-2075.1982.tb00016.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES