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

H. Kadokura, K. Yoda, S. Watanabe, Y. Kikuchi, G. Tamura, M. Yamasaki

研究成果: Article査読

12 被引用数 (Scopus)

抄録

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.

本文言語English
ページ(範囲)163-169
ページ数7
ジャーナルApplied Microbiology and Biotechnology
41
2
DOI
出版ステータスPublished - 1994 4 1
外部発表はい

ASJC Scopus subject areas

  • バイオテクノロジー
  • 応用微生物学とバイオテクノロジー

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