| Summary: | 博士 === 國立中興大學 === 生物科技學研究所 === 92 === Oil bodies of sesame seeds comprise a triacylglycerol matrix, which is surrounded by a monolayer of phospholipids embedded mostly with structural proteins, oleosins. Similar to native oil bodies in vivo and in vitro, reconstituted oil bodies of the same composition maintained as discrete particles of 0.3-3.5 m. Comparable thermostability and structural stability were observed in reconstituted oil bodies as well as purified ones. The sizes of reconstituted oil bodies displayed a normal distribution with an average size proportional to the ratio of triacylglycerols to oil-body proteins. Both thermostability and structural stability of reconstituted oil bodies decreased as their sizes increased, and vice versa. These results suggest that the size of oil bodies is controlled by the availability of triacylglycerols and oil-body proteins during seed maturation, and restricted to a narrow range due to a compromise between minimizing usage of oil-body proteins and stabilizing the organelle effectively. An expression/purification system was developed using artificial oil bodies as carriers for producing recombinant proteins. A target protein, green fluorescent protein (GFP), was firstly expressed in Escherichia coli as an insoluble recombinant protein fused to oleosin, a unique structural protein of seed oil bodies, by a linker sequence susceptible to factor Xa cleavage. Artificial oil bodies were constituted with triacylglycerol, phospholipid, and the insoluble recombinant protein, oleosin-Xa-GFP. After centrifugation, the oleosin-fused GFP was exclusively found on the surface of artificial oil bodies presumably with correct folding to emit fluorescence under excitation. Proteolytic cleavage with factor Xa separated soluble GFP from oleosins that were embedded in the artificial oil bodies; thus after re-centrifugation, GFP of high yield and purity was harvested simply by concentrating the supernatant. Cystatin, a cysteine protease inhibitor, was similarly produced by this system using papain, an inexpensive cysteine protease, in proteolytic cleavage to replace the expensive factor Xa.
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