Skip to main content
Log in

Enhancement of protein secretion by optimizing protein synthesis: isolation and characterization of Escherichia coli mutants with increased secretion ability of alkaline phosphatase

  • Biotechnology
  • Original Paper
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Overexpression of the Escherichia coli phoA gene, coding for alkaline phosphatase, on multicopy plasmids caused the accumulation of the precursor form of alkaline phosphatase. The cells lost their viability by a half-life of 60 min and exhibited high sensitivity to 1% sodium dodecyl sulfate (SDS), suggesting that the assembly of the surface proteins were affected by overexpression of the phoA gene. From the cells exhibiting resistance to 1% SDS, we obtained 20 mutants that secrete more alkaline phosphatase into the periplasmic space. Three representatives of the mutants accumulated no precursor molecules and secreted alkaline phosphatase by five- to six-fold that of the wild-type cells carrying multicopy phoA. In all of the three mutants, the amount of phoA transcripts were two to four times less than those in the wild-type cells, indicating that the ability to secrete a large amount of alkaline phosphatase was conferred by decreasing the synthetic rates of the phoA gene product. When the promoter of phoA was replaced with the tacI promoter and the expression level of the phoA gene was regulated with isopropyl-1-thio-β-d-galactoside, the secretion of alkaline phosphatase into the periplasm decreased as the synthetic rate of the phoA gene product increased over a threshold. All these results indicate that overproduction of the phoA gene product causes defects in the secretion of alkaline phosphatase and that the regulation of the expression level is essential for efficient translocation.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Ames GF-L, Prody C, Kutsu S (1984) Simple, rapid, and quantitative release of periplasmic proteins by chloroform. J Bacteriol 160:1181–1183

    CAS  PubMed  PubMed Central  Google Scholar 

  • Bushman FD, Ptashne M (1988) Turning λ Cro into a transcriptional activator. Cell 54:191–197

    Article  CAS  Google Scholar 

  • De Boer HA, Comstock LJ, Vassar M (1983) The tac promoter: a functional hybrid derived from the trp and lac promoters. Proc Natl Acad Sci USA 80:21–25

    Article  Google Scholar 

  • Freudl R, Schwarz H, Stierhof YD, Gamon K, Hindennach I, Henning U (1986) An outer membrane protein (OmpA) of Escherichia coli K-12 undergoes a conformational change during export. J Biol Chem 261:11355–11361

    CAS  PubMed  Google Scholar 

  • Freudl R, Schwarz H, Kramps S, Hindennach I, Henning U (1988) Dihydrofolate reductase (mouse) and β-galactosidase (Escherichia coli) can be translocated across the plasma membrane of E. coli. J Biol Chem 263:17084–17091

    CAS  PubMed  Google Scholar 

  • Kadokura H, Yoda K, Imai M, Yamasaki M (1990) Production and secretion in Escherichia coli of hepatitis B virus Pre-S2 antigen as fusion proteins with β-lactamase. Appl Environ Microbiol 56:2742–2747

    CAS  PubMed  PubMed Central  Google Scholar 

  • Kusukawa N, Yura T, Ueguchi C, Akiyama Y, Ito K (1989) Effects of mutations in heat-shock genes groES and groEL on protein export in Escherichia coli. EMBO J 8:3517–3521

    Article  CAS  Google Scholar 

  • Kreuzar K, Pratt C, Torriani A (1975) Genetic analysis of regulatory mutants of alkaline phosphatase of E. coli. Genetics 81:459–468

    Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  Google Scholar 

  • Lee C, Beckwith J (1986) Suppression of growth and protein secretion defects in Escherichia coli secA mutants by decreasing protein synthesis. J Bacteriol 166:878–883

    Article  CAS  Google Scholar 

  • Matsuyama S, Fujita Y, Mizushima S (1993) SecD is involved in the release of translocated secretory proteins from the cytoplasmic membrane of Escherichia coli. EMBO J 12:265–270

    Article  CAS  Google Scholar 

  • Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

    Google Scholar 

  • Neidhardt FC, Bloch PL, Smith DF (1974) Culture medium for enterobacteria. J Bacteriol 119:736–747

    CAS  PubMed  PubMed Central  Google Scholar 

  • Oka T, Sakamoto S, Miyoshi K, Fuwa T, Yoda K, Yamasaki M, Tamura G, Miyake T (1985) Synthesis and secretion of human growth factor by Escherichia coli. Proc Natl Acad Sci USA 82:7212–7216

    Article  CAS  Google Scholar 

  • Pages J-M, Anba J, Bernadac A, Shinagawa H, Nakata A, Lazdunski C (1984) Normal precursors of periplasmic protiens accumalated in the cytoplasm are not exported post-translationally in Escherichia coli. Eur J Biochem 143:499–505

    Article  CAS  Google Scholar 

  • Reid TW, Wilson IB (1971) E. coli alkaline phosphatse. In: Boyer PD (ed) The enzymes, vol 4. Academic Press, New York, pp 373–415

    Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

    Google Scholar 

  • Shiba K, Ito K, Yura T (1984) Mutation that suppresses the protein export defect of the secY mutation and causes cold-sensitive growth of Escherichia coli. J Bacteriol 160:696–701

    CAS  PubMed  PubMed Central  Google Scholar 

  • Wanner BL (1987) Phosphate regulation of gene expression in Escherichia coli. In: Neidhardt FC, Ingraham J, Low KB, Magasanik B, Schaechter M, Umbarger HE (eds) Escherichia coli and Salmonella typhimurium; cellular and molecular biology. American Society for Microbiology, Washington, D.C., pp 1326–1333

    Google Scholar 

  • Yamada M, Makino K, Amemura M, Shinagawa H, Nakata A (1989) Regulation of the phosphate regulon of Escherichia coli; analysis of mutant phoB and phoR genes causing different phenotypes. J Bacteriol 171:5601–5606

    Article  CAS  Google Scholar 

  • Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119

    Article  CAS  Google Scholar 

  • Yoda K, Kikuchi Y, Yamasaki M, Tamura G (1980) Cloning of the structural gene of alkaline phosphatase of Escherichia coli K12. Agric Biol Chem 44:1213–1214

    CAS  Google Scholar 

  • Yoda K, Tachibana K, Watanabe S, Yamane K, Yamasaki M, Tamura G (1987) Secretion to periplasm of foreign proteins in Escherichia coli by the aid of the phoA-derived secretion vector Psi. In: Torriani-Gorini A, Rothman FG, Silver S, Wright A, Yagil E (eds) Phosphate metabolism and cellular regulation in microorganisms. American Society for Microbiology, Washington, D.C., pp 99–104

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kadokura, H., Yoda, K., Watanabe, S. et al. Enhancement of protein secretion by optimizing protein synthesis: isolation and characterization of Escherichia coli mutants with increased secretion ability of alkaline phosphatase. Appl Microbiol Biotechnol 41, 163–169 (1994). https://doi.org/10.1007/BF00186954

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00186954

Keywords

Navigation