| Summary: | 博士 === 中興大學 === 生物科技學研究所 === 99 === Plant seeds store neutral lipids (mainly triacylglycerols, TAGs) in an organelle called oil body and use lipids as the energy source for plant germination and subsequent seedling growth. An oil body averaging 0.5-2 um in diameter is made by a matrix of TAGs surrounded by a monolayer of phospholipids (PLs) which anchored with oil body proteins on the surface. There are at least three types of oil body related proteins contain oleosin, caleosin, and steroleosin. Oil bodies maintain their stability as a consequence of electronegative repulsion and steric hindrance provided by oil body proteins on the surface of oil bodies. In vitro, it is possible to reconstitute artificial oil bodies (AOBs) with three essential components, TAGs, PLs and oil body proteins via sonication. Furthermore, AOBs can be constituted with oleosin or caleosin alone but not steroleosin. Several applications based on artificial oil bodies have been developed, including a protein expression/purification system, a drug delivery system, and a technique of enzyme immobilization.
In this dissertation, the stability and application of artificial oil bodies constituted with recombinant caleosins had been studied and performed. Caleosin is one of oil body associated protein has been proposed that have three structural domains with extrusive N- and C-terminal domains and a central hydrophobic domain for anchoring to the surface of seed oil bodies. In addition, the central hydrophobic domain can be divided into an amphiphatic a-helix and a proline-knot subdomain. It has been demonstrated that stable artificial oil bodies were successfully constituted with recombinant caleosin over-expressed in Escherichia coli. In first chapter, the key ability of central domain to constitute and stabilize AOBs was evaluated by recombinant caleosins with their central domain partly or entirely deleted. The stability of artificial oil bodies was examined to show that slightly or severely reduced when the amphiphatic a-helix or proline-knot subdomain in the hydrophobic domain of caleosin was truncated. Furthermore, deletion of the entire central hydrophobic domain substantially increased the solubility of the recombinant caleosin, leading to a complete loss of its capability to stabilize artificial oil bodies. To evaluate the substitution of the hydrophobic domain, a novel protein engineered with the hydrophobic domain of caleosin replaced by that of oleosin, the abundant structural protein of seed oil bodies, could stabilize the artificial oil bodies as effectively as caleosin or oleosin. The success of the novel protein, Cal-Ole-Cal raise the possibility that to engineer different kinds of oil body proteins for diverse AOBs applications. In the second chapter, AOB technique was applied to generate antibodies against small molecules via animal immunization. A series of recombinant caleosins, mutated with extra Lys residues and over-expressed in Escherichia coli, were used as carrier proteins to render biotin and EGCG as model haptens on the surface of artificial oil bodies for the production of anti-hapten antibodies. Biotinylation levels of the recombinant caleosins were step-wisely elevated as the number of extra Lys residues increased, and the exact biotinylated Lys residues were identified by mass spectrometric analysis. Polyclonal antibodies against biotin and EGCG were successfully generated in rats injected with artificial oil bodies constituted with each of the biotinylated and EGCG conjugated caleosins. Moreover, those generated via the biotinylated caleosins with 8 or more extra Lys residues no longer recognized caleosin. It appears that engineered Lys-rich caleosins are suitable carrier proteins for the production of antibodies against small molecules.
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