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 (-18R-->W; 117R-->W) were expressed in Escherichia coli JM109 with isopropyl-beta-D-thio-galactoside (IPTG) induction. Premature cystatin S with the original signal [-20MARPLCTLLLLMATLAGALA] was processed and a large amount of the mature form was produced. A mutation (-18R-->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 periplasmic fraction was prepared by cold osmotic-shock treatment and the expressed cystatins were detected using anti-cystatin S antibody. Recombinant cystatin S and its mutant (117R-->W) were purified from the periplasmic fractions with an ion exchange column of DEAE-cellulose. The amino (N-) terminal 10 residues of recombinant cystatin S was sequenced to be SSSKEENRII-, which is exactly identical to that of the authentic mature cystatin S. Recombinant cystatin S and the mutant showed virtually the same inhibitory properties for ficin, papain and cathepsin B as the native cystatin S and its monophosphorylated form. The inhibitory activity of the both recombinant cystatins for cathepsin C was weaker than those of the native cystatin S and phosphorylated cystatin S.
Saitoh
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E
Department of Oral Biochemistry, Nippon Dental University School of Dentistry at Niigata.
Isemura
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S
D000595:Amino Acid Sequence; D001120:Arginine / Q000235:genetics; D001483:Base Sequence; D015891:Cystatins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D055333:Salivary Cystatins; D014364:Tryptophan / Q000235:genetics
C525171:CST1 protein, human; C525173:CST4 protein, human; D015891:Cystatins; D018076:DNA, Complementary; D011994:Recombinant Proteins; D055333:Salivary Cystatins; D014364:Tryptophan; D001120:Arginine