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

Dalboge, H; Bech Jensen, E; Tottrup, H; Grubb, Anders LU ; Abrahamson, Magnus LU ; Olafsson, I and Carlsen, S (1989) In Gene 79(2). p.325-332
Abstract
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... (More)
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. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
periplasm, cysteine proteinase inhibitor, γ-trace, Recombinant DNA, phage γ, promoter, OmpA signal peptide
in
Gene
volume
79
issue
2
pages
325 - 332
publisher
Elsevier
external identifiers
  • scopus:0024407065
ISSN
1879-0038
DOI
10.1016/0378-1119(89)90214-X
language
English
LU publication?
yes
id
9b8adde0-f0ae-4f42-9d3b-8b55b83ccfa7 (old id 1104865)
date added to LUP
2008-08-06 16:19:02
date last changed
2017-07-30 04:27:37
@article{9b8adde0-f0ae-4f42-9d3b-8b55b83ccfa7,
  abstract     = {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.},
  author       = {Dalboge, H and Bech Jensen, E and Tottrup, H and Grubb, Anders and Abrahamson, Magnus and Olafsson, I and Carlsen, S},
  issn         = {1879-0038},
  keyword      = {periplasm,cysteine proteinase inhibitor,γ-trace,Recombinant DNA,phage γ,promoter,OmpA signal peptide},
  language     = {eng},
  number       = {2},
  pages        = {325--332},
  publisher    = {Elsevier},
  series       = {Gene},
  title        = {High-level expression of active human cystatin C in Escherichia coli},
  url          = {http://dx.doi.org/10.1016/0378-1119(89)90214-X},
  volume       = {79},
  year         = {1989},
}