Molecular cloning, sequencing, expression of Chinese sturgeon cystatin in yeast Pichia pastoris and its proteinase inhibitory activity.
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 pPICZalphaA 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.
Bai
Junjie
J
Pearl River Fisheries Research Institute, CAFS Key Laboratory of Tropical & Subtropical Fish Breeding & Cultivation of CAFS, Guangzhou 510380, PR China. jjbai@163.net
Ma
Dongmei
D
Lao
Haihua
H
Jian
Qing
Q
Ye
Xing
X
Luo
Jianren
J
Xong
Xiaoyong
X
Li
Yinghua
Y
Liang
Xufang
X
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002401:Cathepsin B / Q000037:antagonists & inhibitors / Q000378:metabolism; D003001:Cloning, Molecular; D015891:Cystatins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D029941:Fish Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D005399:Fishes / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010206:Papain / Q000037:antagonists & inhibitors / Q000378:metabolism; D010843:Pichia / Q000235:genetics*; D011480:Protease Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D015891:Cystatins; D018076:DNA, Complementary; D029941:Fish Proteins; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D002401:Cathepsin B; D010206:Papain