Molecular cloning, sequencing, expression of Chinese sturgeon cystatin in yeast Pichia pastoris and its proteinase inhibitory activity

• Published in: Journal of biotechnology 2006-09, Vol.125 (2), p.231-241
• Main Authors: Bai, Junjie, Ma, Dongmei, Lao, Haihua, Jian, Qing, Ye, Xing, Luo, Jianren, Xong, Xiaoyong, Li, Yinghua, Liang, Xufang
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Biological and medical sciences; Biotechnology; Cathepsin B - antagonists & inhibitors; Cathepsin B - metabolism; Chinese sturgeon ( Acipenser sinensis); Cloning, Molecular; Cystatin cDNA; Cystatins - genetics; Cystatins - metabolism; Cystatins - pharmacology; DNA, Complementary - chemistry; DNA, Complementary - genetics; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; Fish Proteins - genetics; Fish Proteins - metabolism; Fish Proteins - pharmacology; Fishes - genetics; Fishes - metabolism; Fundamental and applied biological sciences. Psychology; Gene Expression - genetics; Hot Temperature; Hydrogen-Ion Concentration; Molecular Sequence Data; Papain - antagonists & inhibitors; Papain - metabolism; Pichia - genetics; Pichia pastoris; Protease Inhibitors - pharmacology; Protease inhibitory activity; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Recombinant Proteins - pharmacology; Sequence Alignment; Sequence Analysis, DNA; Sequence Homology, Amino Acid
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2006.02.023

Cystatin, a superfamily of cysteine proteinase inhibitor of cathepsins and other cysteine proteinases, is widely distributed in animal tissues and body fluids. Although considerable attention has been given to mammalian and avian cystatins, little is known about cystatins from other vertebrates. In this study, a cDNA coding for Chinese sturgeon ( Acipenser sinensis) cystatin was isolated and characterized. The corresponding mature cystatin peptide cDNA is 336 nucleotides long and encodes a protein of 112 amino acids. Sequence comparison showed that the cloned cystatin was a homolog of the mammalian Family II cystatin. The cystatin cDNA of Chinese sturgeon was subcloned into yeast expression vector pPICZαA and transformed into Pichia pastoris GS115 strain. After methanol induction, SDS-PAGE analysis of the culture supernatant indicated that the yield of recombinant cystatin was about 215 mg/l medium supernatant in shaking-flask fermentation medium, accounting for 73.6% of the total supernatant secreted proteins. Our data also showed that the recombinant cystatin is active in inhibiting the protease activity of papain and cathepsin B. Heat stability of the recombinant cystatin was also measured.