Molecular cloning, sequencing, expression of Chinese sturgeon cystatin in yeast Pichia pastoris and its proteinase inhibitory activity.
Identifieur interne : 000168 ( Ncbi/Checkpoint ); précédent : 000167; suivant : 000169Molecular cloning, sequencing, expression of Chinese sturgeon cystatin in yeast Pichia pastoris and its proteinase inhibitory activity.
Auteurs : Junjie Bai [République populaire de Chine] ; Dongmei Ma ; Haihua Lao ; Qing Jian ; Xing Ye ; Jianren Luo ; Xiaoyong Xong ; Yinghua Li ; Xufang LiangSource :
- Journal of biotechnology [ 0168-1656 ] ; 2006.
English descriptors
- KwdEn :
- Amino Acid Sequence, Animals, Base Sequence, Cathepsin B (antagonists & inhibitors), Cathepsin B (metabolism), Cloning, Molecular, 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), Gene Expression (genetics), Hot Temperature, Hydrogen-Ion Concentration, Molecular Sequence Data, Papain (antagonists & inhibitors), Papain (metabolism), Pichia (genetics), Protease Inhibitors (pharmacology), Recombinant Proteins (isolation & purification), Recombinant Proteins (metabolism), Recombinant Proteins (pharmacology), Sequence Alignment, Sequence Analysis, DNA, Sequence Homology, Amino Acid.
- MESH :
- chemical , antagonists & inhibitors : Cathepsin B, Papain.
- chemical , chemistry : DNA, Complementary.
- chemical , genetics : Cystatins, DNA, Complementary, Fish Proteins.
- chemical , isolation & purification : Recombinant Proteins.
- chemical , metabolism : Cathepsin B, Cystatins, Fish Proteins, Papain, Recombinant Proteins.
- chemical , pharmacology : Cystatins, Fish Proteins, Protease Inhibitors, Recombinant Proteins.
- genetics : Fishes, Gene Expression, Pichia.
- metabolism : Fishes.
- Amino Acid Sequence, Animals, Base Sequence, Cloning, Molecular, Electrophoresis, Polyacrylamide Gel, Enzyme Stability, Hot Temperature, Hydrogen-Ion Concentration, Molecular Sequence Data, Sequence Alignment, Sequence Analysis, DNA, Sequence Homology, Amino Acid.
Abstract
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.
DOI: 10.1016/j.jbiotec.2006.02.023
PubMed: 16713004
Affiliations:
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<term>Base Sequence</term>
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<term>Cathepsin B (metabolism)</term>
<term>Cloning, Molecular</term>
<term>Cystatins (genetics)</term>
<term>Cystatins (metabolism)</term>
<term>Cystatins (pharmacology)</term>
<term>DNA, Complementary (chemistry)</term>
<term>DNA, Complementary (genetics)</term>
<term>Electrophoresis, Polyacrylamide Gel</term>
<term>Enzyme Stability</term>
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<term>Fish Proteins (metabolism)</term>
<term>Fish Proteins (pharmacology)</term>
<term>Fishes (genetics)</term>
<term>Fishes (metabolism)</term>
<term>Gene Expression (genetics)</term>
<term>Hot Temperature</term>
<term>Hydrogen-Ion Concentration</term>
<term>Molecular Sequence Data</term>
<term>Papain (antagonists & inhibitors)</term>
<term>Papain (metabolism)</term>
<term>Pichia (genetics)</term>
<term>Protease Inhibitors (pharmacology)</term>
<term>Recombinant Proteins (isolation & purification)</term>
<term>Recombinant Proteins (metabolism)</term>
<term>Recombinant Proteins (pharmacology)</term>
<term>Sequence Alignment</term>
<term>Sequence Analysis, DNA</term>
<term>Sequence Homology, Amino Acid</term>
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<term>Papain</term>
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<term>Fish Proteins</term>
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<term>Cystatins</term>
<term>Fish Proteins</term>
<term>Papain</term>
<term>Recombinant Proteins</term>
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<keywords scheme="MESH" type="chemical" qualifier="pharmacology" xml:lang="en"><term>Cystatins</term>
<term>Fish Proteins</term>
<term>Protease Inhibitors</term>
<term>Recombinant Proteins</term>
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<term>Base Sequence</term>
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<term>Electrophoresis, Polyacrylamide Gel</term>
<term>Enzyme Stability</term>
<term>Hot Temperature</term>
<term>Hydrogen-Ion Concentration</term>
<term>Molecular Sequence Data</term>
<term>Sequence Alignment</term>
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<front><div type="abstract" xml:lang="en">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.</div>
</front>
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