Cloning, Functional Expression, and Characterization of Cystatin in Sesame Seed
A cDNA fragment encoding cystatin, a cysteine protease inhibitor, was obtained from maturing sesame seeds. The clone was constructed in a nonfusion or fusion vector and then overexpressed in Escherichia coli. The recombinant cystatins were found in the soluble fraction of cell extract and were demon...
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| Veröffentlicht in: | Journal of agricultural and food chemistry 2004-03, Vol.52 (5), p.1350-1356 |
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| container_title | Journal of agricultural and food chemistry |
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| creator | Shyu, Douglas J. H Chou, Wing-Ming Yiu, Tien-Joung Lin, Coney P. C Tzen, Jason T. C |
| description | A cDNA fragment encoding cystatin, a cysteine protease inhibitor, was obtained from maturing sesame seeds. The clone was constructed in a nonfusion or fusion vector and then overexpressed in Escherichia coli. The recombinant cystatins were found in the soluble fraction of cell extract and were demonstrated to be functionally active in a reverse zymographic assay. The corresponding endogenous 22 kDa cystatin of low abundance in mature seeds was purified to homogeneity via a papain-coupling affinity column and confirmed by western blotting with antibodies against the recombinant cystatin. Both endogenous and recombinant cystatin proteins showed effective inhibitory activities against papain with K i values of 7.89 × 10-8 M and 2.77 × 10-8 M, respectively. Immunodetection indicated that cystatin was specifically expressed in maturing seeds and rapidly degraded in germination. Accordingly, zymographic and inhibition analyses showed that sesame cystatin could not inhibit the de novo synthesized proteases in germinating seeds. It is suggested that sesame cystatin may play a role in the regulation of endogenous cysteine proteases during seed maturation and germination. Keywords: Cystatin; cysteine protease inhibitor; phytocystatin; seed; sesame |
| doi_str_mv | 10.1021/jf034989v |
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H ; Chou, Wing-Ming ; Yiu, Tien-Joung ; Lin, Coney P. C ; Tzen, Jason T. C</creator><creatorcontrib>Shyu, Douglas J. H ; Chou, Wing-Ming ; Yiu, Tien-Joung ; Lin, Coney P. C ; Tzen, Jason T. C</creatorcontrib><description>A cDNA fragment encoding cystatin, a cysteine protease inhibitor, was obtained from maturing sesame seeds. The clone was constructed in a nonfusion or fusion vector and then overexpressed in Escherichia coli. The recombinant cystatins were found in the soluble fraction of cell extract and were demonstrated to be functionally active in a reverse zymographic assay. The corresponding endogenous 22 kDa cystatin of low abundance in mature seeds was purified to homogeneity via a papain-coupling affinity column and confirmed by western blotting with antibodies against the recombinant cystatin. Both endogenous and recombinant cystatin proteins showed effective inhibitory activities against papain with K i values of 7.89 × 10-8 M and 2.77 × 10-8 M, respectively. Immunodetection indicated that cystatin was specifically expressed in maturing seeds and rapidly degraded in germination. Accordingly, zymographic and inhibition analyses showed that sesame cystatin could not inhibit the de novo synthesized proteases in germinating seeds. It is suggested that sesame cystatin may play a role in the regulation of endogenous cysteine proteases during seed maturation and germination. Keywords: Cystatin; cysteine protease inhibitor; phytocystatin; seed; sesame</description><identifier>ISSN: 0021-8561</identifier><identifier>EISSN: 1520-5118</identifier><identifier>DOI: 10.1021/jf034989v</identifier><identifier>PMID: 14995145</identifier><identifier>CODEN: JAFCAU</identifier><language>eng</language><publisher>Washington, DC: American Chemical Society</publisher><subject>Agronomy. Soil science and plant productions ; Amino Acid Sequence ; Biological and medical sciences ; Carrier Proteins - antagonists & inhibitors ; Cloning, Molecular ; Cystatins - chemistry ; Cystatins - genetics ; Cystatins - pharmacology ; DNA, Complementary - isolation & purification ; DNA, Plant - chemistry ; DNA, Plant - isolation & purification ; Economic plant physiology ; Enzyme Inhibitors - pharmacology ; Fundamental and applied biological sciences. Psychology ; Gene Expression ; Molecular Sequence Data ; Morphology. Anatomy. Histology. Cytology ; Seeds - chemistry ; Sequence Alignment ; Sesamum - chemistry</subject><ispartof>Journal of agricultural and food chemistry, 2004-03, Vol.52 (5), p.1350-1356</ispartof><rights>Copyright © 2004 American Chemical Society</rights><rights>2004 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-a379t-83553b2962de1f1dc9880b7c395fcb7e38a6776195930bbb7bdf7861ae50506a3</citedby><cites>FETCH-LOGICAL-a379t-83553b2962de1f1dc9880b7c395fcb7e38a6776195930bbb7bdf7861ae50506a3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://pubs.acs.org/doi/pdf/10.1021/jf034989v$$EPDF$$P50$$Gacs$$H</linktopdf><linktohtml>$$Uhttps://pubs.acs.org/doi/10.1021/jf034989v$$EHTML$$P50$$Gacs$$H</linktohtml><link.rule.ids>314,780,784,2765,27076,27924,27925,56738,56788</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=15561255$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/14995145$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Shyu, Douglas J. H</creatorcontrib><creatorcontrib>Chou, Wing-Ming</creatorcontrib><creatorcontrib>Yiu, Tien-Joung</creatorcontrib><creatorcontrib>Lin, Coney P. C</creatorcontrib><creatorcontrib>Tzen, Jason T. C</creatorcontrib><title>Cloning, Functional Expression, and Characterization of Cystatin in Sesame Seed</title><title>Journal of agricultural and food chemistry</title><addtitle>J. Agric. Food Chem</addtitle><description>A cDNA fragment encoding cystatin, a cysteine protease inhibitor, was obtained from maturing sesame seeds. The clone was constructed in a nonfusion or fusion vector and then overexpressed in Escherichia coli. The recombinant cystatins were found in the soluble fraction of cell extract and were demonstrated to be functionally active in a reverse zymographic assay. The corresponding endogenous 22 kDa cystatin of low abundance in mature seeds was purified to homogeneity via a papain-coupling affinity column and confirmed by western blotting with antibodies against the recombinant cystatin. Both endogenous and recombinant cystatin proteins showed effective inhibitory activities against papain with K i values of 7.89 × 10-8 M and 2.77 × 10-8 M, respectively. Immunodetection indicated that cystatin was specifically expressed in maturing seeds and rapidly degraded in germination. Accordingly, zymographic and inhibition analyses showed that sesame cystatin could not inhibit the de novo synthesized proteases in germinating seeds. It is suggested that sesame cystatin may play a role in the regulation of endogenous cysteine proteases during seed maturation and germination. Keywords: Cystatin; cysteine protease inhibitor; phytocystatin; seed; sesame</description><subject>Agronomy. Soil science and plant productions</subject><subject>Amino Acid Sequence</subject><subject>Biological and medical sciences</subject><subject>Carrier Proteins - antagonists & inhibitors</subject><subject>Cloning, Molecular</subject><subject>Cystatins - chemistry</subject><subject>Cystatins - genetics</subject><subject>Cystatins - pharmacology</subject><subject>DNA, Complementary - isolation & purification</subject><subject>DNA, Plant - chemistry</subject><subject>DNA, Plant - isolation & purification</subject><subject>Economic plant physiology</subject><subject>Enzyme Inhibitors - pharmacology</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Gene Expression</subject><subject>Molecular Sequence Data</subject><subject>Morphology. Anatomy. Histology. Cytology</subject><subject>Seeds - chemistry</subject><subject>Sequence Alignment</subject><subject>Sesamum - chemistry</subject><issn>0021-8561</issn><issn>1520-5118</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2004</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpt0d9r2zAQB3BRNta028P-geGXFQp1J1nRr8fitssgNKVJ6aM4y_LmzLFTnV2a_vVTSGheBoLjuA-H-B4hXxm9ZDRjP5YV5WOjzcsRGTGR0VQwpj-QEY3DVAvJjskJ4pJSqoWin8gxGxsj2FiMyCxvurZuf18kt0Pr-rproUluXtfBI8bmIoG2TPI_EMD1PtRvsCVJVyX5BvvYtEl8c4-w8rH48jP5WEGD_su-npLH25tFPkmns5-_8qtpClyZPtVcCF5kRmalZxUrndGaFspxIypXKM81SKUkM8JwWhSFKspKacnACyqoBH5KznZ716F7Hjz2dlWj800Dre8GtIpJY8ZKRni-gy50iMFXdh3qFYSNZdRu07Pv6UX7bb90KFa-PMh9XBF83wNAB00VoHU1HpyIWWdi69Kdq7H3r-9zCH-tVFwJu7if2-nd4ok_0Gs7OewFh3bZDSFeAf_zwX9OYZGN</recordid><startdate>20040310</startdate><enddate>20040310</enddate><creator>Shyu, Douglas J. H</creator><creator>Chou, Wing-Ming</creator><creator>Yiu, Tien-Joung</creator><creator>Lin, Coney P. C</creator><creator>Tzen, Jason T. C</creator><general>American Chemical Society</general><scope>BSCLL</scope><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>20040310</creationdate><title>Cloning, Functional Expression, and Characterization of Cystatin in Sesame Seed</title><author>Shyu, Douglas J. H ; Chou, Wing-Ming ; Yiu, Tien-Joung ; Lin, Coney P. C ; Tzen, Jason T. C</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a379t-83553b2962de1f1dc9880b7c395fcb7e38a6776195930bbb7bdf7861ae50506a3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2004</creationdate><topic>Agronomy. Soil science and plant productions</topic><topic>Amino Acid Sequence</topic><topic>Biological and medical sciences</topic><topic>Carrier Proteins - antagonists & inhibitors</topic><topic>Cloning, Molecular</topic><topic>Cystatins - chemistry</topic><topic>Cystatins - genetics</topic><topic>Cystatins - pharmacology</topic><topic>DNA, Complementary - isolation & purification</topic><topic>DNA, Plant - chemistry</topic><topic>DNA, Plant - isolation & purification</topic><topic>Economic plant physiology</topic><topic>Enzyme Inhibitors - pharmacology</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Gene Expression</topic><topic>Molecular Sequence Data</topic><topic>Morphology. Anatomy. Histology. Cytology</topic><topic>Seeds - chemistry</topic><topic>Sequence Alignment</topic><topic>Sesamum - chemistry</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Shyu, Douglas J. H</creatorcontrib><creatorcontrib>Chou, Wing-Ming</creatorcontrib><creatorcontrib>Yiu, Tien-Joung</creatorcontrib><creatorcontrib>Lin, Coney P. C</creatorcontrib><creatorcontrib>Tzen, Jason T. C</creatorcontrib><collection>Istex</collection><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Journal of agricultural and food chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Shyu, Douglas J. H</au><au>Chou, Wing-Ming</au><au>Yiu, Tien-Joung</au><au>Lin, Coney P. C</au><au>Tzen, Jason T. C</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Cloning, Functional Expression, and Characterization of Cystatin in Sesame Seed</atitle><jtitle>Journal of agricultural and food chemistry</jtitle><addtitle>J. Agric. Food Chem</addtitle><date>2004-03-10</date><risdate>2004</risdate><volume>52</volume><issue>5</issue><spage>1350</spage><epage>1356</epage><pages>1350-1356</pages><issn>0021-8561</issn><eissn>1520-5118</eissn><coden>JAFCAU</coden><abstract>A cDNA fragment encoding cystatin, a cysteine protease inhibitor, was obtained from maturing sesame seeds. The clone was constructed in a nonfusion or fusion vector and then overexpressed in Escherichia coli. The recombinant cystatins were found in the soluble fraction of cell extract and were demonstrated to be functionally active in a reverse zymographic assay. The corresponding endogenous 22 kDa cystatin of low abundance in mature seeds was purified to homogeneity via a papain-coupling affinity column and confirmed by western blotting with antibodies against the recombinant cystatin. Both endogenous and recombinant cystatin proteins showed effective inhibitory activities against papain with K i values of 7.89 × 10-8 M and 2.77 × 10-8 M, respectively. Immunodetection indicated that cystatin was specifically expressed in maturing seeds and rapidly degraded in germination. Accordingly, zymographic and inhibition analyses showed that sesame cystatin could not inhibit the de novo synthesized proteases in germinating seeds. It is suggested that sesame cystatin may play a role in the regulation of endogenous cysteine proteases during seed maturation and germination. Keywords: Cystatin; cysteine protease inhibitor; phytocystatin; seed; sesame</abstract><cop>Washington, DC</cop><pub>American Chemical Society</pub><pmid>14995145</pmid><doi>10.1021/jf034989v</doi><tpages>7</tpages></addata></record> |
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| subjects | Agronomy. Soil science and plant productions Amino Acid Sequence Biological and medical sciences Carrier Proteins - antagonists & inhibitors Cloning, Molecular Cystatins - chemistry Cystatins - genetics Cystatins - pharmacology DNA, Complementary - isolation & purification DNA, Plant - chemistry DNA, Plant - isolation & purification Economic plant physiology Enzyme Inhibitors - pharmacology Fundamental and applied biological sciences. Psychology Gene Expression Molecular Sequence Data Morphology. Anatomy. Histology. Cytology Seeds - chemistry Sequence Alignment Sesamum - chemistry |
| title | Cloning, Functional Expression, and Characterization of Cystatin in Sesame Seed |
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