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

H Dalboge, E Bech Jensen, H Tottrup, Anders Grubb, Magnus Abrahamson, I Olafsson, S Carlsen

Forskningsoutput: TidskriftsbidragArtikel i vetenskaplig tidskriftPeer review

Sammanfattning

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.
Originalspråkengelska
Sidor (från-till)325-332
TidskriftGene
Volym79
Nummer2
DOI
StatusPublished - 1989

Ämnesklassifikation (UKÄ)

  • Farmakologi och toxikologi
  • Läkemedelskemi

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