AFFINITY PURIFICATION AND ELIMINATION OF METHIONINE OXIDATION IN RECOMBINANT HUMAN CYSTATIN-C

Recombinant human cystatin C (cC), a cysteine protease inhibitor, cont ained methionine sulfoxide [Met(O)] residues when expressed in Escheri chia coli under aerobic conditions or upon allowing osmotic shock solu tions from anaerobically grown cultures to warm to room temperature, O xidation occurred in the periplasmic space or intracellularly during a erobic expression, Both Met14 and Met41 were subject to oxidation, as determined by NMR spectroscopy and mass spectrometry. Oxidation of Met 110 was not observed, Growth under anaerobic conditions and modified p urification procedures prevented oxidation. Through the use of a new f orm of affinity purification, cC was purified to >99% in one step on E -64-papain-Sepharose (E-64 is ne-2-carbonyl)-L-leucyl]amino]-4-guanidi nobutane), with elution with sodium trichloroacetate. The dissociation equilibrium constants (K-d) for the interaction of unoxidized cC, (Me t(O)14)cC, and (Met(O)41)cC with S-(N-ethylsuccinimidyl)papain were ex perimentally 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 struc ture of the protease-binding region of mono-oxidized cC's was unchange d. 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 e arlier upon analytical affinity chromatography. (C) 1997 Academic Pres s.