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研究生:黃琳懿
論文名稱:稻米非專一性脂質運輸蛋白的結構穩定性研究:苯丙胺酸36及雙硫鍵C11-C25的重要性
論文名稱(外文):A Study of Stabilization Factors of Rice Non-specific lipid transfer protein 2 : The Importance of the —CXC-motif and C11-C25 disulfide bond
指導教授:呂平江
指導教授(外文):Ping-Chiang Lyu
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生物技術研究所
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:2003
畢業學年度:91
語文別:中文
論文頁數:72
中文關鍵詞:脂質運輸蛋白結構穩定
外文關鍵詞:lipid transfer proteinstructureCXC motif
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稻米非專一性脂質運輸蛋白是一個小分子量的鹼性蛋白質,有八個高度保留的雙胱胺酸,並且形成四對雙硫鍵。非專一性脂質運輸蛋白的功能是在膜間來往運輸脂質及跟植物防禦有關。非專一性脂質運輸蛋白可依照其分子量分成兩大群,即9k Da的非專一性脂質運輸蛋白1跟7k Da的非專一性脂質運輸蛋白2。這兩群蛋白質的結構都是由四個α-螺旋和一個可移動的C端尾巴所構成。雙硫鍵的配對在這兩群蛋白質中有明顯的不同,而且這個不同是發生在CXC motif 的地方。在非專一性脂質運輸蛋質1中,由於CXC中間的殘基是親水性的,因此這個殘基會面對蛋白質的外圍;而在非專一性脂質運輸蛋白質2中,CXC中間的殘基換成疏水性的因此會被包埋在裡面。這個中間的殘基很可能對於非專一性脂質運輸蛋白的雙硫鍵配對扮演一個蠻重要的角色。
在本論文中,一個在大腸桿菌內的表現系統已被建立完成,可以用來表現野生型和突變株的非專一性脂質運輸蛋白。重組而成的非專一性脂質運輸蛋白在經過檢測後發現與純化而得的蛋白質不論在結構上或是功能上都是一樣的。我們使用單點突變的方法產生F36N和C11A/C25A兩株蛋白質突變種,想知道這幾個殘基在蛋白質結構及功能上的重要性。所得到的結果證明了在36位置的苯丙胺酸的確對結構有重要的影響性,因為這個突變種喪失了很大一部分的α-螺旋。但是將這個殘基取代成天冬醯胺酸後是否就會改變非專一性脂質運輸蛋白的雙硫鍵配對則需要進一步的確認。另一株蛋白質突變種則間接證明了,C11-C25這對雙硫鍵對於非專一性脂質運輸蛋白二級結構的產生及穩定佔有一定的地位。脂質結合的實驗顯現這兩個蛋白質突變種的脂質結合能力已經大大的下降,原因可能是來自於經過突變過後蛋白質本身結構的改變。

Plant non-specific lipid transfer proteins (ns-LTPs) are small, basic proteins which are characterized by eight strictly conserved cysteines forming four disulfide bridges. Ns-LTPs have in vitro lipid transfer and binding ability and seem to play a role in plant defense. Two main groups of ns-LTPs, ns-LTP1 and ns-LTP2, have been identified with molecular weight of about 9 and 7kDa, respectively. Ns-LTP1 and ns-LTP2 share similar structures composed of four α-helices and flexible C-terminal tail. An interesting difference between ns-LTP1 and ns-LTP2 is the mismatched disulfide linkage in CXC motif. In ns-LTP1, “X” possesses hydrophilic residue projecting outside whereas hydrophobic residue burring inside in ns-LTP2. The “X” may act as a key residue determining the disulfide bond pattern.
In this study, an expression system in E. coli was established for the production of ns-LTP2 wild-type and mutants. Recombinant ns-LTP2 is confirmed to be structural and functional identical with purified ns-LTP2. Two mutants were created for the study of structure/function analysis. Phe36 replacement of Asn was introduced for the proof of the hypothesis that the central residue in CXC motif may govern the cysteine pairing and influence the overall fold of the protein. Whether the disulfide bond pattern changed still needs determination. However, the replacement changed the conformation of ns-LTP2. Another mutant, C11A/C25A was introduced for the verifying of the stability caused by disulfide bond. The breakage of this linkage reduced the α-helix content. Both of the mutants displayed a decrease of lipid binding ability which may due to the conformation change brought by mutation.

CHAPTER ONE INTRODUCTION 3
1.1 PLANT NON-SPECIFIC LIPID TRANSFER PROTEIN 3
1.2 PROPOSED BIOLOGICAL ROLES 5
1.3 STRUCTURE PARTICULARITY 7
1.4 RECOMBINANT NS-LTP2 9
1.5 E. COLI EXPRESSION SYSTEM 12
1.6 VECTORS AND HOST CELL USAGE 14
CHAPTER TWO MATERIALS AND METHODS 18
2.1 MATERIALS 18
2.2 CLONING OF LTP2 GENE INTO RECIPIENT VECTOR 18
2.3 EXPRESSION OF NS-LTP2 19
2.4 PURIFICATION OF FUSION NS-LTP2 FROM BACTERIA CULTURE 19
2.5 REMOVAL OF FUSION PARTNER TRX 20
2.6 HPLC ANALYSIS 20
2.7 POLYMERASE CHAIN REACTION 21
2.9 LIGATION 22
2.10 TRANSFORMATION WITH ELECTROPORATION 23
2.11 SDS-PAGE 23
2.12 AGAROSE GEL ELECTROPHOSPHORESIS 24
2.13 EXTRACTION OF PLASMID DNA 24
2.14 PREPARATION OF ELECTRO-COMPETENT CELLS 24
2.15 MASS ANALYSIS 25
2.16 CIRCULAR DICHROISM 25
2.17 FLUORESCENCE SPECTROSCOPY 25
CHAPTER THREE RESULTS 27
3.1 CLONING OF NS-LTP2 INTO VARIOUS VECTORS AND PRODUCTION OF NS-LTP2 IN E. COLI 27
3.2 PURIFICATION OF WILD-TYPE RECOMBINANT NS-LTP2 29
3.3 CONSTRUCTION AND PRODUCTION OF NS-LTP2 MUTANTS IN E. COLI 32
3.4 PURIFICATION OF NS-LTP2 MUTANTS 33
3.5 CHARACTERIZATION OF NS-LTP2 WILD-TYPE AND MUTANTS 34
CHAPTER FOUR DISCUSSION 59
4.1 NS-LTP WITH DISULFIDE BONDS 59
4.2 THE IMPORTANCE OF PHE36 60
4.3 HYDROPHOBIC CORE NEIGHBORING CXC MOTIF IN NS-LTP1 63
4.4 ABOUT C11A-C25A MUTANT 64
CONCLUSION 66
FURTHER WORK 67
APPENDIX 68
REFERENCE 70

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