PRODUCTION OF HUMAN SALIVARY TYPE CYSTEINE PROTEINASE-INHIBITORS (CYSTATINS) BY AN ESCHERICHIA-COLI SYSTEM AND PARTIAL CHARACTERIZATION OF RECOMBINANT CYSTATIN-S AND ITS MUTANT ((117)ARGININE-]TRYPTOPHAN)

The cDNAs encoding the precursors of cystatin SN, cystatin S, and two mutants of cystatin S ((-18)R-->W; (117)R-->W) were expressed in Esche richia coli JM109 with isopropyl-beta-D-thiogalactoside (IPTG) inducti on. Premature cystatin S with the original signal [(-20)MARPLCTLLLLMAT LAGALA] was processed and a large amount of the mature form was produc ed. A mutation ((-18)R-->W) in the signal reduced its accumulation in periplasmic space remarkably. The amount of cystatin SN accumulated in the periplasm was slightly smaller than that of cystatin S. The perip lasmic fraction was prepared by cold osmotic-shock treatment and the e xpressed cystatins were detected using anti-cystatin S antibody. Recom binant cystatin S and its mutant ((117)R-->W) were purified from the p eriplasmic fractions with an ion exchange column of DEAE-cellulose. Th e amino (N-) terminal 10 residues of recombinant cystatin S was sequen ced to be SSSKEENRII-, which is exactly identical to that of the authe ntic mature cystatin S. Recombinant cystatin S and the mutant showed v irtually the same inhibitory properties for ficin, papain and cathepsi n B as the native cystatin S and its monophosphorylated form. The inhi bitory activity of the both recombinant cystatins for cathepsin C was weaker than those of the native cystatin S and phosphorylated cystatin S.