title: Enhancement of proteinase inhibitory activity of recombinant human cystatin C using random-centroid optimization.
abstract: We had previously written a random-centroid optimization computer program for genetics (RCG) to optimize protein engineering, which was successfully applied to modify single site of the 16 amino acid residues at the active site of B. stearothermophilys neutral protease for improving thermostability [J. Agric. Food Chem., 46 (1998) 1655]. The same program was applied in this study to double-site mutation of the entire sequence of human cystatin C (HCC) with 120 residues for improving its protease inhibitory activity. The RCG program selected two sites simultaneously and amino acid residues to replace the sites selected in the sequence in order to find the best papain-inhibitory activity and stability of the protease inhibitor. Twenty-three double mutants and twenty-two single mutants were expressed by Pichia pastoris. Of the total 45 mutants, G12W/H86V mutant showed a 5-fold increase in the bioactivity over the recombinant wild-type (WT) cystatin. Also, P13F mutant exhibited a half-life temperature (T1/2) 5.2 degrees C higher than 68.2 degrees C of WT in addition to a 56% greater papain inhibitory activity. Mutation for diminishing beta-sheet content reduced polymerization of cystatin C, thus improving papain-inhibitory activity. The approach using RCG was able to improve the functional properties of cystatin by least relying on the prior knowledge of its molecular structure.
authors.affiliation: Food, Nutrition, and Health, University of British Columbia, 6650 NW Marine Drive, Vancouver, BC, Canada V6T 1Z4.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Ogawa
authors.forename: Soichiro
authors.initials: S
authors.lastname: Nakamura
authors.forename: Christine H
authors.initials: CH
authors.lastname: Scaman
authors.forename: Hao
authors.initials: H
authors.lastname: Jing
authors.forename: David D
authors.initials: DD
authors.lastname: Kitts
authors.forename: Jingle
authors.initials: J
authors.lastname: Dou
authors.forename: Shuryo
authors.initials: S
authors.lastname: Nakai
mesh_terms: D000818:Animals; D002645:Chickens; D055316:Cystatin C; D015891:Cystatins / Q000235:genetics / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors; D019281:Dimerization; D006801:Humans; D010206:Papain / Q000037:antagonists & inhibitors; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012984:Software
chemical_list: C525030:CST3 protein, human; D055316:Cystatin C; D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D011993:Recombinant Fusion Proteins; D010206:Papain