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Affinity purification and elimination of methionine oxidation in recombinant human cystatin C

Berti, Paul J; Ekiel, Irena; Lindahl, Peter; Abrahamson, Magnus LU and Storer, Andrew C (1997) In Protein Expression and Purification 11(1). p.111-118
Abstract
Recombinant human cystatin C (cC), a cysteine protease inhibitor, contained methionine sulfoxide [Met(O)] residues when expressed in Escherichia coli under aerobic conditions or upon allowing osmotic shock solutions from anaerobically grown cultures to warm to room temperature. Oxidation occurred in the periplasmic space or intracellularly during aerobic expression. Both Met14 and Met41 were subject to oxidation, as determined by NMR spectroscopy and mass spectrometry. Oxidation of Met110 was not observed. Growth under anaerobic conditions and modified purification procedures prevented oxidation. Through the use of a new form of affinity purification, cC was purified to > 99% in one step on E-64-papain-Sepharose (E-64 is... (More)
Recombinant human cystatin C (cC), a cysteine protease inhibitor, contained methionine sulfoxide [Met(O)] residues when expressed in Escherichia coli under aerobic conditions or upon allowing osmotic shock solutions from anaerobically grown cultures to warm to room temperature. Oxidation occurred in the periplasmic space or intracellularly during aerobic expression. Both Met14 and Met41 were subject to oxidation, as determined by NMR spectroscopy and mass spectrometry. Oxidation of Met110 was not observed. Growth under anaerobic conditions and modified purification procedures prevented oxidation. Through the use of a new form of affinity purification, cC was purified to > 99% in one step on E-64-papain-Sepharose (E-64 is 1-[N-[(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]amino]-4-g uanidinobutane), with elution with sodium trichloroacetate. The dissociation equilibrium constants (Kd) for the interaction of unoxidized cC, (Met(O)14)cC, and (Met(O)41)cC with S-(N-ethylsuccinimidyl)papain were experimentally identical: 1.8 (+/-0.2) x 10(-7), 1.6 (+/-0.2) x 10(-7), and 1.4 (+/-0.5) x 10(-7) M, respectively. This implies that the structure of the protease-binding region of mono-oxidized cC's was unchanged. The NMR observation of small, localized conformational changes was consistent with this. (Met(O)14)cC and (Met(O)14,Met(O)41)cC eluted earlier upon analytical affinity chromatography. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Protein Expression and Purification
volume
11
issue
1
pages
111 - 118
publisher
Academic Press
external identifiers
  • pmid:9325146
  • scopus:0031260036
ISSN
1046-5928
DOI
10.1006/prep.1997.0763
language
English
LU publication?
yes
id
e5bf98b4-f77b-4a9c-baff-b3951a5e0785 (old id 1111438)
date added to LUP
2008-07-17 13:32:26
date last changed
2017-03-05 03:25:02
@article{e5bf98b4-f77b-4a9c-baff-b3951a5e0785,
  abstract     = {Recombinant human cystatin C (cC), a cysteine protease inhibitor, contained methionine sulfoxide [Met(O)] residues when expressed in Escherichia coli under aerobic conditions or upon allowing osmotic shock solutions from anaerobically grown cultures to warm to room temperature. Oxidation occurred in the periplasmic space or intracellularly during aerobic expression. Both Met14 and Met41 were subject to oxidation, as determined by NMR spectroscopy and mass spectrometry. Oxidation of Met110 was not observed. Growth under anaerobic conditions and modified purification procedures prevented oxidation. Through the use of a new form of affinity purification, cC was purified to > 99% in one step on E-64-papain-Sepharose (E-64 is 1-[N-[(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]amino]-4-g uanidinobutane), with elution with sodium trichloroacetate. The dissociation equilibrium constants (Kd) for the interaction of unoxidized cC, (Met(O)14)cC, and (Met(O)41)cC with S-(N-ethylsuccinimidyl)papain were experimentally identical: 1.8 (+/-0.2) x 10(-7), 1.6 (+/-0.2) x 10(-7), and 1.4 (+/-0.5) x 10(-7) M, respectively. This implies that the structure of the protease-binding region of mono-oxidized cC's was unchanged. The NMR observation of small, localized conformational changes was consistent with this. (Met(O)14)cC and (Met(O)14,Met(O)41)cC eluted earlier upon analytical affinity chromatography.},
  author       = {Berti, Paul J and Ekiel, Irena and Lindahl, Peter and Abrahamson, Magnus and Storer, Andrew C},
  issn         = {1046-5928},
  language     = {eng},
  number       = {1},
  pages        = {111--118},
  publisher    = {Academic Press},
  series       = {Protein Expression and Purification},
  title        = {Affinity purification and elimination of methionine oxidation in recombinant human cystatin C},
  url          = {http://dx.doi.org/10.1006/prep.1997.0763},
  volume       = {11},
  year         = {1997},
}