Affinity purification and elimination of methionine oxidation in recombinant human cystatin C.
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.
Berti
P J
PJ
Pharmaceutical Biotechnology Sector, Biotechnology Research Institute, National Research Council of Canada, Montréal, Quebec, Canada.
Ekiel
I
I
Lindahl
P
P
Abrahamson
M
M
Storer
A C
AC
D002846:Chromatography, Affinity; D055316:Cystatin C; D015891:Cystatins / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008715:Methionine / Q000378:metabolism*; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
C525030:CST3 protein, human; D055316:Cystatin C; D015891:Cystatins; D011994:Recombinant Proteins; D008715:Methionine