Rainbow trout (Oncorhynchus mykiss) cystatin C: expression in Escherichia coli and properties of the recombinant protease inhibitor.
Cystatins are a superfamily of low Ki cysteine proteinase inhibitors found in both plants and animals. Cystatin C, a secreted molecule of this family, is of interest from biochemical and evolutionary points of view, and also has biotechnological applications. Recently we cloned and sequenced the cDNA for rainbow trout (Oncorhynchus mykiss) cystatin C [Li et al., 1998. Molecular cloning, sequence analysis and expression distribution of rainbow trout (Oncorhynchus mykiss) cystatin C. Comp. Biochem. Phys. B 121, 135-143]. To explore the relationship between protein structure and function of trout cystatin C, we established a bacterial system for expression of the protein. Trout cystatin C expressed in the cytoplasm of bacterial cells did not have detectable protease inhibitory activity. Activity was regained by Ni-NTA chromatography under denaturing conditions followed by dialysis-based refolding. Titration of purified cystatin C preparations with papain indicated that approximately 20% of the total protein had been converted to the active form after one refolding cycle. Expression levels were 3-5 mg/l. The protease-inhibitory properties of recombinant trout cystatin C were similar to those of human and chicken cystatin C derived from biological sources and recombinant cystatin C derived from rat and mouse genes. The Ki for papain was 1.2 x 10(-15) M, exhibiting the high affinity binding unique to this family of protease inhibitors.
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Department of Genetics, Washington University School of Medicine, St Louis, MO 63110, USA.
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Seymour
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Barnes
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D000818:Animals; D055316:Cystatin C; D015891:Cystatins / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology*; D015853:Cysteine Proteinase Inhibitors / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D051379:Mice; D017686:Oncorhynchus mykiss; D010206:Papain / Q000037:antagonists & inhibitors; D011401:Promoter Regions, Genetic; D015202:Protein Engineering / Q000379:methods; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology
C525030:CST3 protein, human; C525031:Cst3 protein, mouse; C525032:Cst3 protein, rat; D055316:Cystatin C; D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D011994:Recombinant Proteins; D010206:Papain