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

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.

• Författare: H Dalboge; E Bech Jensen; H Tottrup; Anders Grubb; Magnus Abrahamson; I Olafsson; S Carlsen
• Avdelning/ar: Avdelningen för klinisk kemi och farmakologi
• Publiceringsår: 1989
• Språk: Engelska
• Sidor: 325-332
• Publikation/Tidskrift/Serie: Gene
• Volym: 79
• Issue: 2
• Länkar: http://dx.doi.org/10.1016/0378-1119(89)90214-X
• Dokumenttyp: Artikel i tidskrift
• Förlag: Elsevier
• Ämne: Pharmacology and Toxicology; Medicinal Chemistry
• Nyckelord: periplasm; cysteine proteinase inhibitor; γ-trace; Recombinant DNA; phage γ; promoter; OmpA signal peptide
• Status: Published
• ISBN/ISSN/Övrigt: ISSN: 1879-0038