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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 orcid ; 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
; ; ; ; ; and
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
2016-04-01 15:34:17
date last changed
2021-06-13 03:34:11
@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}},
  keywords     = {{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}},
  doi          = {{10.1016/0378-1119(89)90214-X}},
  volume       = {{79}},
  year         = {{1989}},
}