Method for bacterial expression and purification of sesame cystatin via artificial oil bodies.
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 55 degrees C 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.
Peng
Chi-Chung
CC
Graduate Institute of Biotechnology, National Chung-Hsing University, Taichung, Taiwan.
Shyu
Douglas J H
DJ
Chou
Wing-Ming
WM
Chen
Ming-Jinn
MJ
Tzen
Jason T C
JT
D015891:Cystatins / Q000235:genetics* / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D015388:Organelles; D010206:Papain / Q000037:antagonists & inhibitors; D010940:Plant Proteins / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D012639:Seeds / Q000737:chemistry / Q000648:ultrastructure*; D031685:Sesamum / Q000737:chemistry*
D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; C110161:oleosin protein, Sesamum indicum; D010206:Papain