Molecular cloning, sequencing, expression of Chinese sturgeon cystatin in yeast Pichia pastoris and its proteinase inhibitory activity
Identifieur interne : 000D56 ( Main/Exploration ); précédent : 000D55; suivant : 000D57Molecular 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 [République populaire de Chine] ; HAIHUA LAO [République populaire de Chine] ; QING JIAN [République populaire de Chine] ; XING YE [République populaire de Chine] ; JIANREN LUO [République populaire de Chine] ; XIAOYONG XONG [République populaire de Chine] ; YINGHUA LI [République populaire de Chine] ; XUFANG LIANG [République populaire de Chine]Source :
- Journal of biotechnology [ 0168-1656 ] ; 2006.
Descripteurs français
- Pascal (Inist)
English descriptors
- KwdEn :
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 pPICZaA 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.
Affiliations:
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Key Laboratory of Tropical & Subtropical Fish Breeding & Cultivation of CAFS</s1><s2>Guangzhou 510380</s2><s3>CHN</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>3 aut.</sZ><sZ>4 aut.</sZ><sZ>5 aut.</sZ><sZ>6 aut.</sZ><sZ>7 aut.</sZ><sZ>8 aut.</sZ></inist:fA14><country>République populaire de Chine</country><placeName><settlement type="city">Jiangmen</settlement><region type="province">Guangdong</region></placeName></affiliation></author><author><name sortKey="Dongmei Ma" sort="Dongmei Ma" uniqKey="Dongmei Ma" last="Dongmei Ma">DONGMEI MA</name><affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Pearl River Fisheries Research Institute, CAFS. Key Laboratory of Tropical & Subtropical Fish Breeding & Cultivation of CAFS</s1><s2>Guangzhou 510380</s2><s3>CHN</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>3 aut.</sZ><sZ>4 aut.</sZ><sZ>5 aut.</sZ><sZ>6 aut.</sZ><sZ>7 aut.</sZ><sZ>8 aut.</sZ></inist:fA14><country>République populaire de Chine</country><placeName><settlement type="city">Jiangmen</settlement><region type="province">Guangdong</region></placeName></affiliation><affiliation wicri:level="1"><inist:fA14 i1="02"><s1>Zhanjiang Ocean University</s1><s2>Zhanjiang 520425</s2><s3>CHN</s3><sZ>2 aut.</sZ></inist:fA14><country>République populaire de Chine</country><wicri:noRegion>Zhanjiang Ocean University</wicri:noRegion></affiliation></author><author><name sortKey="Haihua Lao" sort="Haihua Lao" uniqKey="Haihua Lao" last="Haihua Lao">HAIHUA LAO</name><affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Pearl River Fisheries Research Institute, CAFS. Key Laboratory of Tropical & Subtropical Fish Breeding & Cultivation of CAFS</s1><s2>Guangzhou 510380</s2><s3>CHN</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>3 aut.</sZ><sZ>4 aut.</sZ><sZ>5 aut.</sZ><sZ>6 aut.</sZ><sZ>7 aut.</sZ><sZ>8 aut.</sZ></inist:fA14><country>République populaire de Chine</country><placeName><settlement type="city">Jiangmen</settlement><region type="province">Guangdong</region></placeName></affiliation></author><author><name sortKey="Qing Jian" sort="Qing Jian" uniqKey="Qing Jian" last="Qing Jian">QING JIAN</name><affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Pearl River Fisheries Research Institute, CAFS. Key Laboratory of Tropical & Subtropical Fish Breeding & Cultivation of CAFS</s1><s2>Guangzhou 510380</s2><s3>CHN</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>3 aut.</sZ><sZ>4 aut.</sZ><sZ>5 aut.</sZ><sZ>6 aut.</sZ><sZ>7 aut.</sZ><sZ>8 aut.</sZ></inist:fA14><country>République populaire de Chine</country><placeName><settlement type="city">Jiangmen</settlement><region type="province">Guangdong</region></placeName></affiliation></author><author><name sortKey="Xing Ye" sort="Xing Ye" uniqKey="Xing Ye" last="Xing Ye">XING YE</name><affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Pearl River Fisheries Research Institute, CAFS. Key Laboratory of Tropical & Subtropical Fish Breeding & Cultivation of CAFS</s1><s2>Guangzhou 510380</s2><s3>CHN</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>3 aut.</sZ><sZ>4 aut.</sZ><sZ>5 aut.</sZ><sZ>6 aut.</sZ><sZ>7 aut.</sZ><sZ>8 aut.</sZ></inist:fA14><country>République populaire de Chine</country><placeName><settlement type="city">Jiangmen</settlement><region type="province">Guangdong</region></placeName></affiliation></author><author><name sortKey="Jianren Luo" sort="Jianren Luo" uniqKey="Jianren Luo" last="Jianren Luo">JIANREN LUO</name><affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Pearl River Fisheries Research Institute, CAFS. Key Laboratory of Tropical & Subtropical Fish Breeding & Cultivation of CAFS</s1><s2>Guangzhou 510380</s2><s3>CHN</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>3 aut.</sZ><sZ>4 aut.</sZ><sZ>5 aut.</sZ><sZ>6 aut.</sZ><sZ>7 aut.</sZ><sZ>8 aut.</sZ></inist:fA14><country>République populaire de Chine</country><placeName><settlement type="city">Jiangmen</settlement><region type="province">Guangdong</region></placeName></affiliation></author><author><name sortKey="Xiaoyong Xong" sort="Xiaoyong Xong" uniqKey="Xiaoyong Xong" last="Xiaoyong Xong">XIAOYONG XONG</name><affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Pearl River Fisheries Research Institute, CAFS. Key Laboratory of Tropical & Subtropical Fish Breeding & Cultivation of CAFS</s1><s2>Guangzhou 510380</s2><s3>CHN</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>3 aut.</sZ><sZ>4 aut.</sZ><sZ>5 aut.</sZ><sZ>6 aut.</sZ><sZ>7 aut.</sZ><sZ>8 aut.</sZ></inist:fA14><country>République populaire de Chine</country><placeName><settlement type="city">Jiangmen</settlement><region type="province">Guangdong</region></placeName></affiliation><affiliation wicri:level="1"><inist:fA14 i1="03"><s1>Fishery college, Shanghai Fisheries University</s1><s2>Shanghai 200090</s2><s3>CHN</s3><sZ>7 aut.</sZ></inist:fA14><country>République populaire de Chine</country><wicri:noRegion>Shanghai 200090</wicri:noRegion></affiliation></author><author><name sortKey="Yinghua Li" sort="Yinghua Li" uniqKey="Yinghua Li" last="Yinghua Li">YINGHUA LI</name><affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Pearl River Fisheries Research Institute, CAFS. Key Laboratory of Tropical & Subtropical Fish Breeding & Cultivation of CAFS</s1><s2>Guangzhou 510380</s2><s3>CHN</s3><sZ>1 aut.</sZ><sZ>2 aut.</sZ><sZ>3 aut.</sZ><sZ>4 aut.</sZ><sZ>5 aut.</sZ><sZ>6 aut.</sZ><sZ>7 aut.</sZ><sZ>8 aut.</sZ></inist:fA14><country>République populaire de Chine</country><placeName><settlement type="city">Jiangmen</settlement><region type="province">Guangdong</region></placeName></affiliation><affiliation wicri:level="1"><inist:fA14 i1="04"><s1>Department of Biotechnology, College of Life Science and Technology, Jinan University</s1><s2>Guangzhou 510632</s2><s3>CHN</s3><sZ>8 aut.</sZ><sZ>9 aut.</sZ></inist:fA14><country>République populaire de Chine</country><placeName><settlement type="city">Jiangmen</settlement><region type="province">Guangdong</region></placeName></affiliation></author><author><name sortKey="Xufang Liang" sort="Xufang Liang" uniqKey="Xufang Liang" last="Xufang Liang">XUFANG LIANG</name><affiliation wicri:level="1"><inist:fA14 i1="04"><s1>Department of Biotechnology, College of Life Science and Technology, Jinan University</s1><s2>Guangzhou 510632</s2><s3>CHN</s3><sZ>8 aut.</sZ><sZ>9 aut.</sZ></inist:fA14><country>République populaire de Chine</country><placeName><settlement type="city">Jiangmen</settlement><region type="province">Guangdong</region></placeName></affiliation></author></analytic><series><title level="j" type="main">Journal of biotechnology</title><title level="j" type="abbreviated">J. biotechnol.</title><idno type="ISSN">0168-1656</idno><imprint><date when="2006">2006</date></imprint></series></biblStruct></sourceDesc><seriesStmt><title level="j" type="main">Journal of biotechnology</title><title level="j" type="abbreviated">J. biotechnol.</title><idno type="ISSN">0168-1656</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Complementary DNA</term><term>Cystatin</term><term>Inhibition</term><term>Molecular cloning</term><term>Peptidases</term><term>Pichia pastoris</term><term>Sequencing</term><term>Yeast</term></keywords><keywords scheme="Pascal" xml:lang="fr"><term>Clonage moléculaire</term><term>Séquençage</term><term>Cystatine</term><term>Levure</term><term>Peptidases</term><term>Pichia pastoris</term><term>Inhibition</term><term>DNA complémentaire</term></keywords></textClass></profileDesc></teiHeader><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 pPICZaA 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></TEI><affiliations><list><country><li>République populaire de Chine</li></country><region><li>Guangdong</li></region><settlement><li>Jiangmen</li></settlement></list><tree><country name="République populaire de Chine"><region name="Guangdong"><name sortKey="Junjie Bai" sort="Junjie Bai" uniqKey="Junjie Bai" last="Junjie Bai">JUNJIE BAI</name></region><name sortKey="Dongmei Ma" sort="Dongmei Ma" uniqKey="Dongmei Ma" last="Dongmei Ma">DONGMEI MA</name><name sortKey="Dongmei Ma" sort="Dongmei Ma" uniqKey="Dongmei Ma" last="Dongmei Ma">DONGMEI MA</name><name sortKey="Haihua Lao" sort="Haihua Lao" uniqKey="Haihua Lao" last="Haihua Lao">HAIHUA LAO</name><name sortKey="Jianren Luo" sort="Jianren Luo" uniqKey="Jianren Luo" last="Jianren Luo">JIANREN LUO</name><name sortKey="Qing Jian" sort="Qing Jian" uniqKey="Qing Jian" last="Qing Jian">QING JIAN</name><name sortKey="Xiaoyong Xong" sort="Xiaoyong Xong" uniqKey="Xiaoyong Xong" last="Xiaoyong Xong">XIAOYONG XONG</name><name sortKey="Xiaoyong Xong" sort="Xiaoyong Xong" uniqKey="Xiaoyong Xong" last="Xiaoyong Xong">XIAOYONG XONG</name><name sortKey="Xing Ye" sort="Xing Ye" uniqKey="Xing Ye" last="Xing Ye">XING YE</name><name sortKey="Xufang Liang" sort="Xufang Liang" uniqKey="Xufang Liang" last="Xufang Liang">XUFANG LIANG</name><name sortKey="Yinghua Li" sort="Yinghua Li" uniqKey="Yinghua Li" last="Yinghua Li">YINGHUA LI</name><name sortKey="Yinghua Li" sort="Yinghua Li" uniqKey="Yinghua Li" last="Yinghua Li">YINGHUA LI</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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