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研究生:季欣文
研究生(外文):Hsin-Wen Chi
論文名稱:馬來蝮蛇蛇毒P48E/R49I/G50L/M52V突變蛋白在大腸桿菌與酵母菌中的表現與特性之研究
論文名稱(外文):Expression and Characterization of P48E/R49I/G50L/M52V Mutant of Rhodostomin in Escherichia coli and Pichia pastoris
指導教授:王惠亮莊偉哲
指導教授(外文):Hui-Liang WangWoei-Jer Chuang
學位類別:碩士
校院名稱:國立高雄師範大學
系所名稱:生物科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:79
中文關鍵詞:馬來蝮蛇蛇毒蛋白突變蛋白構築純化定性分析表現大腸桿菌酵母菌
外文關鍵詞:RhodostominP48E/R49I/G50L/M52V Mutant of RhodostominConstructionPurificationCharacterizationExpressionEscherichia coliPichia pastoris
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中文摘要
結合蛋白(integrin)是一群由a、b異質雙體所形成之細胞表面接受器的大家族,它們的功能與細胞的黏著、移動和訊息傳遞有關。不同的結合蛋白會辨認不同的胞外間質(extracellular matrix),且將近一半的結合蛋白是會辨識在它們所黏結的胞外間質上的RGD(Arg-Gly-Asp)序列。去結合蛋白 (disintegrin) 是一群由蛇毒中所純化出來的蛋白,可以破壞結合蛋白的功能。而馬來蝮蛇蛇毒蛋白(Rhodostomin, Rho)即是去結合蛋白的一種,它是由馬來蝮蛇(Calloselasma rhodostoma)中所純化出來的蛋白,共有68個胺基酸、其中有6對雙硫鍵,而RGD序列則是位於第49-51的胺基酸序列。為了研究結合蛋白其選擇性所需要的識別序列,本實驗以Rhodostomin為立體支架的材料,設計兩個突變引子EILDV-1及EILDV-2針對RGD及RGD附近的胺基酸序列作突變成EILDV,並將此EILDV突變基因構築於pET21a質體 (E. coli) 及pPICZaA 質體 (Pichia pastoris) 上,並且誘發突變蛋白的表現。利用SDS-PAGE分析檢測此突變蛋白。進一步將在Pichia pastoris系統上所表現的Rho EILDV突變蛋白以鎳離子螯合親合性色層分析法(Ni2+-chelating chromatography)及逆相高效能液相層析法(RP-HPLC ; Reverse Phase-High Performance Liquid Chromomatography) 純化此蛋白,純化後的Rho EILDV突變蛋白的產率 (yield) 為5 mg/L。而在定性分析方面,純化後的Rho EILDV突變蛋白送質譜儀 (Mass) 確定分子量為8430.3 Da,與電腦分析所得蛋白質序列分子量的理論值吻合;並利用核磁共振 (NMR spectroscopy )做結構的測定,在pH 6.0的NOESY圖譜中,與未突變的Rhodostomin 蛋白比較,兩者在骨幹 (backbone) 結構是相似的,在支鏈 (side chain) 上因突變而有所不同,從NOESY圖譜中所得到明顯的點證明其結構的正確。

Abstract
Integrins are a family of a、b heterodimeric cell surface receptors that involve in cellular adhesion, migration and signal transduction. Nearly half of integrins recognize the RGD(Arg-Gly-Asp)sequence in the extracellular matrix. Disintegrins are found in snake venoms, and they have high potency in inhibiting the function of integrins. Rhodostomin (Rho), a RGD sequence-containing disintegrin, is a snake venom protein isolated from Agkistodon rhodostoma, a Malayan pit viper. Rho consists of 68 amino acids including six disulfide bonds and a RGD sequence at positions of 49-51. In this study, the amino acids in the RGD and adjacent to RGD region of Rhodostomin were mutated to EILDV, and the EILDV mutant was constructed into E.coli and P. pastoris expression systems. The Rho EILDV mutant was also expressed in E.coli as well as P. pastoris, and the proteins were analyzed by SDS-PAGE. The protein was purified by Ni2+-chelating chromatography and Reverse Phase-High Performance Liquid Chromomatography (RP-HPLC), and the yield of the Rho EILDV mutant protein was 5 mg/L. The molecular weight of Rho EILDV mutant determined by Mass spectrometry was 8430.3 Da that was the same as the result of the prediction from the computer. According to the NOESY spectra of Rho EILDV mutant at pH 6.0 determined by NMR, there was no difference between wild type Rho and the Rho EILDV mutant and the folding of Rho EILDV mutant was correct.

目 錄
中文摘要…………………………………………………………………………………I
英文摘要………………………………………………………………………………..III
壹、 前言……………………………………………………………………………….1
一、 結合蛋白 (integrin) 之介紹……………......................................................1
二、 去結合蛋白 (disintegrin) 之介紹…………….............................................3
三、 馬來蝮蛇去結合蛋白 ( rhodostomin; 簡稱Rho)之介紹………………….7
四、 論文研究動機及內容之介紹………………………………………………...9
貳、 材料與方法……………………………………………………………………….12
一、 實驗菌株與質體……………………………………………………………..12
二、 Rhodostomin 基因之來源…………………………………………………...12
三、 Rhodostomin P48E/R49I/G50L/M52V………………………………………14
突變株的重組基因(EILDV)之構築
四、 Rhodostomin P48E/R49I/G50L/M52V………………………………………27
突變株的重組基因(EILDV)蛋白質
誘發表現及純化
五、質譜(Mass)與核磁共振 (NMR) 之分析…………………………………..32
參、 實驗結果…………………………………………………………………………..34
一、 Rhodostomin P48E/R49I/G50L/M52V突變基因之構築……………………34
二、 Rhodostomin P48E/R49I/G50L/M52V突變蛋白之表現及純化……………48
三、 Rhodostomin P48E/R49I/G50L/M52V突變蛋白的
質譜分析(Mass) 與核磁共振 (NMR)…………………………………………54
肆、 討論………………………………………………………………………………..59
伍、 參考文獻…………………………………………………………………………..63
附錄……………………………………………………………………………………….69

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