Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/50412
標題: Method for bacterial expression and purification of sesame cystatin via artificial oil bodies
作者: Peng, C.C.
曾志正
Shyu, D.J.H.
Chou, W.M.
Chen, M.J.
Tzen, J.T.C.
關鍵字: artificial oil bodies
cystatin
papain
protease inhibitor
oleosin
cysteine-proteinase-inhibitor
chicken cystatin
escherichia-coli
animal cystatins
seed
plant
oleosin
cloning
caleosin
enzymes
期刊/報告no:: Journal of Agricultural and Food Chemistry, Volume 52, Issue 10, Page(s) 3115-3119.
摘要: A method was developed for production of Sesame cystatin, a thermostable cysteine protease inhibitor. Sesame cystatin was first expressed in Escherichia coli as an insoluble recombinant protein fused to oleosin, a unique structural protein of seed oil bodies, by a short hydrophilic linker peptide. Stable artificial oil bodies were constituted with triacylglycerol, phospholipid, and the insoluble oleosin-cystatin fusion protein. After centrifugation, the oleosin-cystatin fusion protein was exclusively found in the artificial oil bodies. Proteolytic cleavage with papain, a cysteine protease effectively inhibited by cystatin, separated soluble cystatin from oleosin that was firmly embedded in the artificial oil bodies. After recentrifugation, papain that coexisted with cystatin in the collected supernatant was denatured by incubating at 55degreesC for 30 min. The insoluble denatured papain was removed by one more centrifugation, and the expressed cystatin of high yield and purity was harvested simply by concentrating the ultimate supernatant. Comparable inhibitory activity toward papain was observed between the expressed cystatin and the native one purified from sesame seeds. This method is presumably applicable to production of other protease inhibitors whose target proteases are economically available.
URI: http://hdl.handle.net/11455/50412
ISSN: 0021-8561
文章連結: http://dx.doi.org/10.1021/jf049849f
Appears in Collections:生物科技學研究所

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