Abstract
We have constructed a secretion vector containing genes that code for the Escherichia coli ompA signal peptide and human growth hormone (hGH). The recombinant fusion protein was expressed in E. coli cells harboring the vector and the correctly processed hGH was secreted into the E. coli periplasm, yielding 10–15 μg hGH/A600 cells in the periplasm. Purified hGH was shown to have the correct amino terminus, and also the correct disulfide bonds and the proper secondary structure. The results indicate that the E. coli periplasm can provide an environment to facilitate efficient disulfide bond formation and proper folding of hGH. The purification and isolation of the recombinant protein are greatly simplified and the processes needed to refold recombinant proteins derived from the E. coli cytoplasm are eliminated.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Schoner, R.G., Ellis, L.F., and Schoner, B.E. 1985. Isolation and purification of protein granules from Escherichia coli cells overproducing bovine growth hormone. Bio/Technology 3:151–154.
Glasbrenner, K. 1986. Technology spurt resolves growth hormone problem, ends shortage. JAMA 255:581–587.
Tokunaga, M., Loranger, J.M., Wolfe, P.B., and Wu, H.C. 1982. Prolipoprotein signal peptidase in Escherichia coli is distinct from M13 procoat protein signal peptidase. J. Biol. Chem. 257:9922–9925.
Wolfe, P.B., Silver, P., and Wickner, W. 1982. The isolation of homogeneous leader peptidase from a strain of Escherichia coli which overproduces the enzyme. J. Biol. Chem. 257:7898–7902.
Ghrayeb, J., Kimura, H., Takahara, M., Hsiung, H., Msaui, Y., and Inouye, M.J. 1984. Secretion cloning vectors in Escherichia coli. The EMBO J. 3:2437–2442.
Takahara, M., Hibler, D.W., Barr, P.J., Gerlt, J.A., and Inouye, M. 1985. The ompA signal peptide directed secretion of staphylococcal nuclease A by Escherichia coli. J. Biol. Chem. 260:2670–2674.
Gray, G.L., Baldridge, J.S., McKeown, K.S., Heyneker, H.L., and Chang, C.N. 1985. Periplasmic production of correctly processed human growth hormone in Escherichia coli: natural and bacterial signal sequences are interchangeable. Gene 39:247–254.
Matteucci, M., and Lipetsky, H. 1986. Alkaline phosphatase fusion: a tag to identify mutations that result in increased expression of secreted human growth hormone from E. coli. Bio/Technology 4:51–55.
Oka, T., Sakamoto, S., Miyoshi, K., Fuwa, T., Yoda, K., Yamasaki, M., Tamura, G., and Miyake, T. 1985. Synthesis and secretion of human epidermal growth factor by Escherichia coli. Proc. Natl. Acad. Sci. USA 82:7212–7216.
Koshland, D., and Botstein, D. 1980. Secretion of beta-lactamase requires the carboxy end of the protein. Cell 20:749–760.
Becker, G.W. and Hsiung, H.M. 1986. Expression, secretion and folding of human growth hormone in Escherichia coli: purification and characterization. FEBS Letters, in press.
Talmadge, K., Stahl, S., and Gilbert, W. 1980. Eukaryotic signal sequence transports insulin antigen in Escherichia coli. Proc. Natl. Acad. Sci. USA 77:3369–3373.
Talmadge, K., Kaufman, J., and Gilbert, W. 1980. Bacteria mature prepoinsulin to proinsulin. Proc. Natl. Acad. Sci. USA, 77:3988–3992.
Emerick, A.W., Bertolani, B.L., Ben-Bassat, A., White, T.J. and Konrad, M.W. 1984. Expression of a β-lactamase preproinsulin fusion protein in Escherichia coli. Bio/Technology 2:165–168.
Tackitt, P.M. and Becker, G.W. Personal communication.
Bewley, T.A., Brovetto-Cruz, J., and Li, C.H. 1969. Human pituitary growth hormone. Physicochemical investigations of the native and reduced-alkylated protein. Biochem. 8:4701–4708.
Narang, S.A., Hsiung, H.M., and Brousseau, R. 1979. Improved phosphotriester methods for the synthesis of gene fragments. Methods in Enzymology 68:90–98.
Martial, J.A., Hallewell, R.A., Baxter, J.D., and Goodman, H.M. 1979. Human growth hormone: complementary DNA and expression in bacteria. Science 205:602–607.
Morinaga, Y., Franceschini, T., Inouye, S., and Inouye, M. 1984. Improvements of oligonucieotide-directed site-specific mutagenesis using double-stranded plasmid DNA. Bio/Technology 2:636–639.
Maxam, A., and Gilbert, W. 1980. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods in Enzymology 65:499–560.
Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of bacteriophage T4. Nature 277:680–685.
Burnette, W.N. 1981. “Western Blotting”: electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal. Biochem. 112:195–203.
Sportsman, J.R. 1986. Manuscript in preparation.
Riggin, R.M. and Becker, G.W. 1986. Manuscript in preparation.
Somack, R. 1980. Complete phenylthiohydantoin amino acid analysis by high-performance liquid chromatography on Ultrasphere-octadecyltrimethyloxysilane. Anal. Biochem. 104:464–468.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Hsiung, H., Mayne, N. & Becker, G. High–Level Expression, Efficient Secretion and Folding of Human Growth Hormone in Escherichia coli. Nat Biotechnol 4, 991–995 (1986). https://doi.org/10.1038/nbt1186-991
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1038/nbt1186-991
This article is cited by
-
Production of human growth hormone in transgenic rice seeds: co-introduction of RNA interference cassette for suppressing the gene expression of endogenous storage proteins
Plant Cell Reports (2012)
-
Overproduction of a recombinant carboxypeptidase inhibitor by optimization of fermentation conditions
Applied Microbiology and Biotechnology (1994)
-
Over-expression of ?-lytic protease and its mutants by recombinantEscherichia coli
Biotechnology Letters (1994)
-
Efficient Bacterial Export of a Eukaryotic Cytoplasmic Cytochrome
Nature Biotechnology (1993)
-
Secretion of a functional Fab fragment in Escherichia coli and the influence of culture conditions
Applied Microbiology and Biotechnology (1992)