Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1985 Dec 16;4(13A):3589–3592. doi: 10.1002/j.1460-2075.1985.tb04121.x

Secretion into the culture medium of a foreign gene product from Escherichia coli: use of the ompF gene for secretion of human beta-endorphin.

K Nagahari, S Kanaya, K Munakata, Y Aoyagi, S Mizushima
PMCID: PMC554701  PMID: 2936602

Abstract

The ompF gene codes for a major outer membrane protein of Escherichia coli. A plasmid was constructed in which the structural gene for human beta-endorphin is preceded by the upstream region of the ompF gene consisting of the promoter region and the coding regions for the signal peptide and the N terminus of the OmpF protein. When the plasmid was introduced into E. coli N99, and OmpF-beta-endorphin fused peptide was synthesized and secreted into the culture medium through both the cytoplasmic and outer membranes. The OmpF signal peptide was cleaved correctly during the secretion, indicating that the export of the fused protein across the cytoplasmic membrane was dependent on the signal peptide. The secretion into the culture medium was apparently selective. Neither beta-lactamase nor alkaline phosphatase (both are periplasmic proteins) appeared in the culture medium in significant amounts. The mode of passage of the fused peptide across the outer membrane is discussed.

Full text

PDF
3592

Selected References

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

  1. Bolivar F., Backman K. Plasmids of Escherichia coli as cloning vectors. Methods Enzymol. 1979;68:245–267. doi: 10.1016/0076-6879(79)68018-7. [DOI] [PubMed] [Google Scholar]
  2. Chan S. J., Weiss J., Konrad M., White T., Bahl C., Yu S. D., Marks D., Steiner D. F. Biosynthesis and periplasmic segregation of human proinsulin in Escherichia coli. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5401–5405. doi: 10.1073/pnas.78.9.5401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Hall M. N., Silhavy T. J. Genetic analysis of the ompB locus in Escherichia coli K-12. J Mol Biol. 1981 Sep 5;151(1):1–15. doi: 10.1016/0022-2836(81)90218-7. [DOI] [PubMed] [Google Scholar]
  4. Inokuchi K., Furukawa H., Nakamura K., Mizushima S. Characterization by deletion mutagenesis in vitro of the promoter region of ompF, a positively regulated gene of Escherichia coli. J Mol Biol. 1984 Sep 25;178(3):653–668. doi: 10.1016/0022-2836(84)90243-2. [DOI] [PubMed] [Google Scholar]
  5. Inokuchi K., Mutoh N., Matsuyama S., Mizushima S. Primary structure of the ompF gene that codes for a major outer membrane protein of Escherichia coli K-12. Nucleic Acids Res. 1982 Nov 11;10(21):6957–6968. doi: 10.1093/nar/10.21.6957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Kudo T., Kato C., Horikoshi K. Excretion of the penicillinase of an alkalophilic Bacillus sp. through the Escherichia coli outer membrane. J Bacteriol. 1983 Nov;156(2):949–951. doi: 10.1128/jb.156.2.949-951.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Mackman N., Holland I. B. Secretion of a 107 K dalton polypeptide into the medium from a haemolytic E. coli K12 strain. Mol Gen Genet. 1984;193(2):312–315. doi: 10.1007/BF00330686. [DOI] [PubMed] [Google Scholar]
  9. Michaelis S., Beckwith J. Mechanism of incorporation of cell envelope proteins in Escherichia coli. Annu Rev Microbiol. 1982;36:435–465. doi: 10.1146/annurev.mi.36.100182.002251. [DOI] [PubMed] [Google Scholar]
  10. Mutoh N., Inokuchi K., Mizushima S. Amino acid sequence of the signal peptide of OmpF, a major outer membrane protein of Escherichia coli. FEBS Lett. 1982 Jan 25;137(2):171–174. doi: 10.1016/0014-5793(82)80341-4. [DOI] [PubMed] [Google Scholar]
  11. Müller D., Hughes C., Goebel W. Relationship between plasmid and chromosomal hemolysin determinants of Escherichia coli. J Bacteriol. 1983 Feb;153(2):846–851. doi: 10.1128/jb.153.2.846-851.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Nakamura Y., Yura T. Evidence for a positive regulation of RNA polymerase synthesis in Escherichia coli. J Mol Biol. 1975 Oct 5;97(4):621–642. doi: 10.1016/s0022-2836(75)80063-5. [DOI] [PubMed] [Google Scholar]
  13. Nakanishi S., Inoue A., Kita T., Nakamura M., Chang A. C., Cohen S. N., Numa S. Nucleotide sequence of cloned cDNA for bovine corticotropin-beta-lipotropin precursor. Nature. 1979 Mar 29;278(5703):423–427. doi: 10.1038/278423a0. [DOI] [PubMed] [Google Scholar]
  14. Nikaido H., Vaara M. Molecular basis of bacterial outer membrane permeability. Microbiol Rev. 1985 Mar;49(1):1–32. doi: 10.1128/mr.49.1.1-32.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Rosenberg M., Ho Y. S., Shatzman A. The use of pKc30 and its derivatives for controlled expression of genes. Methods Enzymol. 1983;101:123–138. doi: 10.1016/0076-6879(83)01009-5. [DOI] [PubMed] [Google Scholar]
  16. Sawai T., Takahashi I., Yamagishi S. Iodometric assay method for beta-lactamase with various beta-lactam antibiotics as substrates. Antimicrob Agents Chemother. 1978 Jun;13(6):910–913. doi: 10.1128/aac.13.6.910. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Takahashi H., Teranishi Y., Nakanishi S., Numa S. Isolation and structural organization of the human corticotropin--beta-lipotropin precursor gene. FEBS Lett. 1981 Nov 30;135(1):97–102. doi: 10.1016/0014-5793(81)80952-0. [DOI] [PubMed] [Google Scholar]
  18. Talmadge K., Stahl S., Gilbert W. Eukaryotic signal sequence transports insulin antigen in Escherichia coli. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3369–3373. doi: 10.1073/pnas.77.6.3369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]

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

RESOURCES