Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1987 May;84(9):2638–2642. doi: 10.1073/pnas.84.9.2638

Expression of a synthetic gene encoding human insulin-like growth factor I in cultured mouse fibroblasts.

M L Bayne, M A Cascieri, B Kelder, J Applebaum, G Chicchi, J A Shapiro, F Pasleau, J J Kopchick
PMCID: PMC304713  PMID: 3033657

Abstract

A synthetic gene encoding human insulin-like growth factor I (hIGF-I) was assembled and inserted into an expression vector containing the cytomegalovirus immediate early (CMV-IE) transcriptional regulatory region and portions of the bovine growth hormone gene. The recombinant plasmid encodes a 97 amino acid fusion protein containing the first 27 amino acids of the bovine growth hormone precursor and the 70 amino acids of hIGF-I. This plasmid, when transiently introduced into cultured mouse fibroblasts, directs synthesis of the fusion protein, subsequent proteolytic removal of the bovine growth hormone signal peptide, and secretion of hIGF-I into the culture medium. Conditioned medium from transfected cells inhibits binding of 125I-labeled IGF-I to type I IGF receptors on human placental membranes and to acid-stable human serum carrier proteins. The recombinant hIGF-I produced is biologically active, as monitored by the stimulation of DNA synthesis in vascular smooth muscle cells.

Full text

PDF
2638

Images in this article

2641Image
on p.2641

Selected References

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

  1. Bell G. I., Gerhard D. S., Fong N. M., Sanchez-Pescador R., Rall L. B. Isolation of the human insulin-like growth factor genes: insulin-like growth factor II and insulin genes are contiguous. Proc Natl Acad Sci U S A. 1985 Oct;82(19):6450–6454. doi: 10.1073/pnas.82.19.6450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blundell T. L., Bedarkar S., Humbel R. E. Tertiary structures, receptor binding, and antigenicity of insulinlike growth factors. Fed Proc. 1983 Jun;42(9):2592–2597. [PubMed] [Google Scholar]
  4. Blundell T. L., Bedarkar S., Rinderknecht E., Humbel R. E. Insulin-like growth factor: a model for tertiary structure accounting for immunoreactivity and receptor binding. Proc Natl Acad Sci U S A. 1978 Jan;75(1):180–184. doi: 10.1073/pnas.75.1.180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Buell G., Schulz M. F., Selzer G., Chollet A., Movva N. R., Semon D., Escanez S., Kawashima E. Optimizing the expression in E. coli of a synthetic gene encoding somatomedin-C (IGF-I). Nucleic Acids Res. 1985 Mar 25;13(6):1923–1938. doi: 10.1093/nar/13.6.1923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cascieri M. A., Chicchi G. G., Hayes N. S., Slater E. E. Stimulation of DNA synthesis in rat A10 vascular smooth muscle cells by threonine-59 insulin-like growth factor I. Circ Res. 1986 Aug;59(2):171–177. doi: 10.1161/01.res.59.2.171. [DOI] [PubMed] [Google Scholar]
  7. Clemmons D. R., Elgin R. G., Han V. K., Casella S. J., D'Ercole A. J., Van Wyk J. J. Cultured fibroblast monolayers secrete a protein that alters the cellular binding of somatomedin-C/insulinlike growth factor I. J Clin Invest. 1986 May;77(5):1548–1556. doi: 10.1172/JCI112470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Copeland K. C., Underwood L. E., Van Wyk J. J. Induction of immunoreactive somatomedin C human serum by growth hormone: dose-response relationships and effect on chromatographic profiles. J Clin Endocrinol Metab. 1980 Apr;50(4):690–697. doi: 10.1210/jcem-50-4-690. [DOI] [PubMed] [Google Scholar]
  9. De Vroede M. A., Tseng L. Y., Katsoyannis P. G., Nissley S. P., Rechler M. M. Modulation of insulinlike growth factor I binding to human fibroblast monolayer cultures by insulinlike growth factor carrier proteins released to the incubation media. J Clin Invest. 1986 Feb;77(2):602–613. doi: 10.1172/JCI112343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Jansen M., van Schaik F. M., Ricker A. T., Bullock B., Woods D. E., Gabbay K. H., Nussbaum A. L., Sussenbach J. S., Van den Brande J. L. Sequence of cDNA encoding human insulin-like growth factor I precursor. Nature. 1983 Dec 8;306(5943):609–611. doi: 10.1038/306609a0. [DOI] [PubMed] [Google Scholar]
  11. Kopchick J. J., Malavarca R. H., Livelli T. J., Leung F. C. Use of avian retroviral-bovine growth hormone DNA recombinants to direct expression of biologically active growth hormone by cultured fibroblasts. DNA. 1985 Feb;4(1):23–31. doi: 10.1089/dna.1985.4.23. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Lo K. M., Jones S. S., Hackett N. R., Khorana H. G. Specific amino acid substitutions in bacterioopsin: Replacement of a restriction fragment in the structural gene by synthetic DNA fragments containing altered codons. Proc Natl Acad Sci U S A. 1984 Apr;81(8):2285–2289. doi: 10.1073/pnas.81.8.2285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Pasleau F., Tocci M. J., Leung F., Kopchick J. J. Growth hormone gene expression in eukaryotic cells directed by the Rous sarcoma virus long terminal repeat or cytomegalovirus immediate-early promoter. Gene. 1985;38(1-3):227–232. doi: 10.1016/0378-1119(85)90221-5. [DOI] [PubMed] [Google Scholar]
  17. Peters M. A., Lau E. P., Snitman D. L., Van Wyk J. J., Underwood L. E., Russell W. E., Svoboda M. E. Expression of a biologically active analogue of somatomedin-C/insulin-like growth factor I. Gene. 1985;35(1-2):83–89. doi: 10.1016/0378-1119(85)90160-x. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Rotwein P., Pollock K. M., Didier D. K., Krivi G. G. Organization and sequence of the human insulin-like growth factor I gene. Alternative RNA processing produces two insulin-like growth factor I precursor peptides. J Biol Chem. 1986 Apr 15;261(11):4828–4832. [PubMed] [Google Scholar]
  20. Rotwein P. Two insulin-like growth factor I messenger RNAs are expressed in human liver. Proc Natl Acad Sci U S A. 1986 Jan;83(1):77–81. doi: 10.1073/pnas.83.1.77. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. Ullrich A., Berman C. H., Dull T. J., Gray A., Lee J. M. Isolation of the human insulin-like growth factor I gene using a single synthetic DNA probe. EMBO J. 1984 Feb;3(2):361–364. doi: 10.1002/j.1460-2075.1984.tb01812.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Woychik R. P., Camper S. A., Lyons R. H., Horowitz S., Goodwin E. C., Rottman F. M. Cloning and nucleotide sequencing of the bovine growth hormone gene. Nucleic Acids Res. 1982 Nov 25;10(22):7197–7210. doi: 10.1093/nar/10.22.7197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES