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High-level expression of active human cystatin C in Escherichia coli

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Author

Summary, in English

Expression of the human cysteine proteinase inhibitor, cystatin C (CysC) in the cytoplasm of Escherichia coli was studied using a cDNA fragment encoding the cysteine proteinase inhibitor controlled by the phage λ pImage /cI857 system. The yield of CysC was low, probably due to proteolytic degradation. By fusing the cysC cDNA to a DNA fragment encoding the signal peptide of the E. coli outer membrane protein A, it was possible to produce a substantial amount of CysC in the periplasm. The processing of the signal peptide was shown to be quantitative and to result in CysC with the correct N-terminal amino acid. Yields higher than 1000 μg CysC/ml can be obtained by initiating the product formation at a moderate temperature (40 °C) late in an optimized fermentation process. A method that gives selective extraction of the periplasmic proteins and at the same time stabilizes CysC has been used.

Publishing year

1989

Language

English

Pages

325-332

Publication/Series

Gene

Volume

79

Issue

2

Document type

Journal article

Publisher

Elsevier

Topic

  • Pharmacology and Toxicology
  • Medicinal Chemistry

Keywords

  • periplasm
  • cysteine proteinase inhibitor
  • γ-trace
  • Recombinant DNA
  • phage γ
  • promoter
  • OmpA signal peptide

Status

Published

ISBN/ISSN/Other

  • ISSN: 1879-0038