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研究生:彭及忠
研究生(外文):Peng Chi-Chung
論文名稱:人造芝麻油體的技術開發及應用
論文名稱(外文):Development and Applications of Artificial Sesame Oil Body
指導教授:曾 志 正
指導教授(外文):Jason T. C. Tzen
學位類別:博士
校院名稱:國立中興大學
系所名稱:生物科技學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:英文
論文頁數:98
中文關鍵詞:三酸甘油酯油體油體膜蛋白
外文關鍵詞:triacylglycerolsoil bodyoleosin
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油體為植物種子內特有胞器,主要儲存三酸甘油酯(triacylglycerols)以提供種子發芽後生長所需能量來源。芝麻油體主要結構為三酸甘油酯包覆於單層磷脂質(phospholipids)內,在磷酯層上則鑲滿了油體膜蛋白(oleosin)及一些微量蛋白質。依植物油體之組成比例將三酸甘油酯、磷脂及油體膜蛋白混合,經超音波震盪後既可組成與天然油體類似的人造油體。若改變三酸甘油酯與蛋白質比例則會形成不同大小的人造油體;三酸甘油酯比例較高形成的人造油體較大但穩定性較差,反之、蛋白質比例較高形成的油體較小但穩定性卻比較好。以此人造油體作為一生物科技平台,用來發展融合蛋白質純化系統的研發應用。以一特殊的綠色螢光蛋白(GFP)做為目標蛋白質,並在兩蛋白質間設計一專一蛋白質水解酵素(Factor Xa)切位,再利用微生物來大量表達此一融合蛋白質後,加入三酸甘油酯和磷脂經超音波震盪形成人造油體,經離心純化人造油體並發現融合蛋白質的另一端GFP可正確呈現綠色螢光於人造油體表面,再以專一蛋白質水解酵素Factor Xa作用將GFP與人造油體分離,並經離心後將油體分離及純化的GFP。另外並以此蛋白質純化系統研發一高效率又經濟的新方法來純化半胱胺酸蛋白酶抑制蛋白質(cystatin)。以較經濟的半胱胺酸蛋白酶取代昂貴的專一性蛋白酶Factor Xa,並成功的純化半胱胺酸蛋白酶抑制蛋白質。
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.
Table of Contents
Chinese Abstract 1
Abstract 2
General Introductuin 4
Reference 15
Figures 20
Chapter 1. Size and Stability of Reconstituted Oil Bodies Are Affected 29
by the Ratio of Triacylglycerols to Oil-Body Proteins
Introduction 30
Materials and methods 33
Results 38
Discussion 42
References 44
Table 48
Figures 49
Chapter 2. A System for Purification of Recombinant Proteins in 59
Escherichia coli via Artificial Oil Bodies Constituted
with Their Oleosin-fused Polypeptides
Introduction 60
Materials and methods 62
Results 65
Discussion 67
References 69
Figure 72
Chapter 3. Method for Bacterial Expression and Purification of 77
Sesame Cystatin via Artificial Oil Bodies
Introduction 78
Materials and methods 80
Results 84
Discussion 86
References 88
Figure 91
Conclusion 96
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