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標題: Cloning, functional expression, and characterization of cystatin in sesame seed
作者: Shyu, D.J.H.
Chou, W.M.
Yiu, T.J.
Lin, C.P.C.
Tzen, J.T.C.
關鍵字: cystatin;cysteine protease inhibitor;phytocystatin;seed;sesame;cysteine proteinase-inhibitor;programmed cell-death;escherichia-coli;oil bodies;animal cystatins;storage proteins;oryzacystatin-i;plants;binding;rice
Project: Journal of Agricultural and Food Chemistry
期刊/報告no:: Journal of Agricultural and Food Chemistry, Volume 52, Issue 5, Page(s) 1350-1356.
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 x 10(-8) M and 2.77 x 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.
ISSN: 0021-8561
DOI: 10.1021/jf034989v
Appears in Collections:生物科技學研究所

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