Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1985 Feb 25;13(4):1151–1162. doi: 10.1093/nar/13.4.1151

Efficient secretion and purification of human insulin-like growth factor I with a gene fusion vector in Staphylococci.

B Nilsson, E Holmgren, S Josephson, S Gatenbeck, L Philipson, M Uhlen
PMCID: PMC341062  PMID: 3889837

Abstract

A novel approach for production of small polypeptides, using a staphylococcal protein A vector, is described. This system is used to express, secrete and purify human insulin-like growth factor I (IGF-I). A fusion protein consisting of protein A and IGF-I is recovered in high yield by passing the culture medium through an IgG affinity column. Using site-specific mutagenesis an acid labile asp-pro cleavage site was introduced at the fusion point between the two proteins. The protein A "tail" can thereby be removed from the affinity purified fusion protein by chemical cleavage releasing biologically active IGF-I molecules.

Full text

PDF
1151

Images in this article

1157Image
on p.1157

Selected References

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

  1. Clemmons D. R., Van Wyk J. J. Somatomedin-C and platelet-derived growth factor stimulate human fibroblast replication. J Cell Physiol. 1981 Mar;106(3):361–367. doi: 10.1002/jcp.1041060305. [DOI] [PubMed] [Google Scholar]
  2. Everett R. D., Chambon P. A rapid and efficient method for region- and strand-specific mutagenesis of cloned DNA. EMBO J. 1982;1(4):433–437. doi: 10.1002/j.1460-2075.1982.tb01187.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Germino J., Bastia D. Rapid purification of a cloned gene product by genetic fusion and site-specific proteolysis. Proc Natl Acad Sci U S A. 1984 Aug;81(15):4692–4696. doi: 10.1073/pnas.81.15.4692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Gillam S., Astell C. R., Smith M. Site-specific mutagenesis using oligodeoxyribonucleotides: isolation of a phenotypically silent phi X174 mutant, with a specific nucleotide deletion, at very high efficiency. Gene. 1980 Dec;12(1-2):129–137. doi: 10.1016/0378-1119(80)90023-2. [DOI] [PubMed] [Google Scholar]
  5. Goeddel D. V., Kleid D. G., Bolivar F., Heyneker H. L., Yansura D. G., Crea R., Hirose T., Kraszewski A., Itakura K., Riggs A. D. Expression in Escherichia coli of chemically synthesized genes for human insulin. Proc Natl Acad Sci U S A. 1979 Jan;76(1):106–110. doi: 10.1073/pnas.76.1.106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hall K., Takano K., Fryklund L. Radioreceptor assay for somatomedin A. J Clin Endocrinol Metab. 1974 Nov;39(5):973–976. doi: 10.1210/jcem-39-5-973. [DOI] [PubMed] [Google Scholar]
  7. Horinouchi S., Weisblum B. Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance. J Bacteriol. 1982 May;150(2):815–825. doi: 10.1128/jb.150.2.815-825.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Itakura K., Hirose T., Crea R., Riggs A. D., Heyneker H. L., Bolivar F., Boyer H. W. Expression in Escherichia coli of a chemically synthesized gene for the hormone somatostatin. Science. 1977 Dec 9;198(4321):1056–1063. doi: 10.1126/science.412251. [DOI] [PubMed] [Google Scholar]
  9. Kudo I., Leineweber M., RajBhandary U. L. Site-specific mutagenesis on cloned DNAs: generation of a mutant of Escherichia coli tyrosine suppressor tRNA in which the sequence G-T-T-C corresponding to the universal G-T-pseudouracil-C sequence of tRNAs is changed to G-A-T-C. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4753–4757. doi: 10.1073/pnas.78.8.4753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Landon Cleavage at aspartyl-prolyl bonds. Methods Enzymol. 1977;47:145–149. doi: 10.1016/0076-6879(77)47017-4. [DOI] [PubMed] [Google Scholar]
  12. Nagai K., Thøgersen H. C. Generation of beta-globin by sequence-specific proteolysis of a hybrid protein produced in Escherichia coli. 1984 Jun 28-Jul 4Nature. 309(5971):810–812. doi: 10.1038/309810a0. [DOI] [PubMed] [Google Scholar]
  13. Nordström K., Lindberg M. Effects of streptomycin and novobiocin on Staphylococcus aureus gene expression. J Bacteriol. 1978 Feb;133(2):614–620. doi: 10.1128/jb.133.2.614-620.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Palva I., Lehtovaara P., Käriäinen L., Sibakov M., Cantell K., Schein C. H., Kashiwagi K., Weissmann C. Secretion of interferon by Bacillus subtilis. Gene. 1983 May-Jun;22(2-3):229–235. doi: 10.1016/0378-1119(83)90107-5. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Schoenle E., Zapf J., Humbel R. E., Froesch E. R. Insulin-like growth factor I stimulates growth in hypophysectomized rats. Nature. 1982 Mar 18;296(5854):252–253. doi: 10.1038/296252a0. [DOI] [PubMed] [Google Scholar]
  17. Uhlén M., Guss B., Nilsson B., Götz F., Lindberg M. Expression of the gene encoding protein A in Staphylococcus aureus and coagulase-negative staphylococci. J Bacteriol. 1984 Aug;159(2):713–719. doi: 10.1128/jb.159.2.713-719.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Zapf J., Schoenle E., Froesch E. R. Insulin-like growth factors I and II: some biological actions and receptor binding characteristics of two purified constituents of nonsuppressible insulin-like activity of human serum. Eur J Biochem. 1978 Jun 15;87(2):285–296. doi: 10.1111/j.1432-1033.1978.tb12377.x. [DOI] [PubMed] [Google Scholar]
  20. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 1983;100:468–500. doi: 10.1016/0076-6879(83)00074-9. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES