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研究生:陳俊同
研究生(外文):Chun-Tung Chen
論文名稱:基因選殖及大量表現根瘤菌之根瘤因子硫酸根轉移
論文名稱(外文):Cloning and Overexpression of Nod Factor Sulfotransferase from Sinorhizobium meliloti
指導教授:林俊宏林俊宏引用關係
指導教授(外文):Chun-Hung Lin
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生化科學研究所
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:2000
畢業學年度:88
語文別:中文
論文頁數:82
中文關鍵詞:Nod Factor SulfotransferaseSinorhizobium melilotiNod FactorCapillary ElectrophoresisPAPS
外文關鍵詞:根瘤因子硫酸根轉移根瘤菌根瘤因子毛細管電泳
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中文摘要
硫酸根轉移是一種酵素的總稱,它會催化反應將硫酸根從3’-phosphoadenosine-5’-phosphosulfate (PAPS) 轉移到受質上而產生帶有硫酸根化合物。帶硫酸根化合物在生理上扮演著重要的角色,例如,毒物的代謝、細胞訊息的傳遞、接合器 (receptor) 接合效果,所以可以藉由合成各種抑制物,來了解硫酸根轉移的作用機制及生理功能。
根瘤化因子在根瘤菌與豆科植物間的共生作用中,扮演著訊息傳遞的功能,豆科植物會分泌類黃酮素刺激根瘤菌,以表現一系列的蛋白質作為訊息傳遞物質─根瘤化因子。根瘤化因子 (Nod factors) 就是一種帶有硫酸根的糖分子,它的結構是由四個或五個GlcNAc,經由β-1、4鍵結而形成的寡醣,且在其還原端的第六個氧上會被修飾而帶有一個硫酸根。根瘤化因子之硫酸根轉移酵素 (Nod factor sulfotransferase),即是負責將相關的醣分子在特定的位置作硫酸根化反應。
本研究論文是針對Nod Sulfotransferase,其基因是從根瘤菌(Sinorhizobium meliloti)中所選殖出,在大腸桿菌 (E. coli) 中表現蛋白質,這個酵素是由248個胺基酸所組成的,分子量約為28,000。Nod Sulfotransferase經大量表現後發現,這酵素很容易聚集,而形成包涵體 (inclusion body)。針對這樣的問題,改善的方式是採取降低誘導的溫度、降低誘導物質IPTG的濃度與選擇不同載體,來提高可溶性蛋白質的量。最佳的結果是選用pET21的載體,以16 ℃及0.01 mM的IPTG誘導蛋白質的表現,再利用鎳金屬離子之親和層析管柱 (nickel column) 來純化蛋白質。
本文藉由毛細管電泳 (Capillary electrophoresis, CE) 來建立新的分析方法,作為此酵素的活性分析方法,因為CE具備有高解析的功能,以取代傳統的同位素標定法。以N,N’,N’’-triacetylchitotriose為受質時,由此分析方法可以得到酵素動力學參數Km 232 µM、Kcat 20 min-1。
Abstract
Sulfotransferases represent a family of enzymes to catalyze the transfer of a sulfate group from 3’-phosphoadenosine-5’-phosphosulfate (PAPS) to an acceptor molecule. Sulfated biomolecules play important roles in many biological processes including detoxification, cell signaling, and modulation of receptor binding. Followed by the discovery and investigations of sulfotransferases, many approaches of the drug design for the inhibition of these therapeutically interesting enzymes have been initiated.
In the symbiotic relationship between Rhizobium bacteria and leguminous host plants, a set of molecular signals are exchanged. Rhizobia have a cluster of nod genes required for the production of the signal molecules, the lipo-oligosaccharide nodulation factors. They are ß-1,4-linked N-acetylglucosamine oligosaccharides with the sulfate at the C-6 position of the reducing sugar. Nod sulfotransferase carries out the sulfation for the modification of the Nod factors.
The nod factor sulfotransferase was cloned from Sinorhizobium meliloti (CCRC 13530) and expressed in E. coli. The enzyme encodes a polypeptide of 248 amino acid residues with a calculated MW 28,585. The expressed protein was found to aggregate easily leading to the formation of inclusion body. To overcome such a problem, induction and culturing conditions were optimized on protein expression. By studying effects of temperatures, IPTG concentrations, and expression vectors, the optimum condition was determined to use the pET21vector and to induce in 0.01 mM IPTG at 16 ℃. The soluble enzyme was purified by nickel ion (Ni2+) affinity chromatography and eluted out from column in 250 mM imidazole.
A new analytical method based on capillary electrophoresis was also developed for the sulfotransferase assay to replace the previous isotope-labeling method. When N,N’,N’’-Tri-N-acetylchitotriose was used as a substrate for kinetic analysis, the parameters, Km and kcat , were determined to be 210 µM and 20 min-1, respectively.
目錄
謝誌………………………………………………………………………Ⅰ
目錄………………………………………………………………………Ⅲ
圖表目錄…………………………………………………………………Ⅴ
縮寫表……………………………………………………………………Ⅶ
中文摘要…………………………………………………………………Ⅷ
英文摘要…………………………………………………………………Ⅹ
第一章、緒論
1.1. 硫酸化醣類的重要性………………………………………………1
1.2. 硫酸根轉移(sulfotransferase)……………………………2
1.3. 硫酸化醣類的合成…………………………………………………5
1.4. 根瘤硫酸根轉移(Nod sulfotransferase) ………………6
1.5. 根瘤硫酸根轉移的基因及表現…………………………………8
1.6. N,N'',N''''-Tri-N-acetylchitotriose-6-sulfate的製備.10
1.7. 酵素活性的分析 …………………………………………………10
1.8. 實驗目的………………………………………………………… 12
第二章、實驗材料與方法
2.1. 菌株與質體………………………………………………………13
2.2. 藥品………………………………………………………………15
2.3. 細菌的培養與生長測定…………………………………………15
2.4. 根瘤菌的培養……………………………………………………15
2.5. 根瘤菌染色體核酸的製備………………………………………16
2.6. 引子(Primer)的設計與聚合鏈鎖反應(PCR)………………17
2.7. 質體DNA的製備…………………………………………………18
2.8. 核酸濃度的定量…………………………………………………19
2.9. 核酸限制酵素切割反應…………………………………………19
2.10. 核酸膠體電泳分析(Gel Electrephoresis of DNA)……20
2.11. 膠體中核酸片段的回收與純化………………………………20
2.12. 核酸的接合實驗(DNA ligation)……………………………21
2.13. 細胞的轉型作用 (Transformation)………………………21
2.14. 基因的篩選………………………………………………………22
2.15. 菌種的保存………………………………………………………23
2.16. 破菌的方法………………………………………………………23
2.17. 蛋白質溶液的定量………………………………………………24
2.18. 蛋白質電泳分析…………………………………………………24
2.19. 西方點墨免疫轉印法 (Western hybridization)………27
2.20. 重組蛋白質的純化………………………………………………28
2.21. 根瘤硫酸根轉移的活性分析方法……………………………29
2.22. 毛細管電泳分析方法………………………………………….30
第三章、實驗結果
3.1. 根瘤硫酸根轉移的基因選殖…………………………………31
3.2. 根瘤硫酸根轉移的活性分析結果……………………………35
3.3. 重組蛋白質根瘤硫酸根轉移的純化…………………………35
3.4. 製備大量帶硫酸根的化合物……………………………………35
3.5. 毛細管電泳分析的結果…………………………………………37
第四章、實驗討論
4.1. 根瘤菌的培養……………………………………………………39
4.2. 根瘤硫酸轉移的基因序列……………………………………40
4.3. 根瘤硫酸轉移的氨基酸序列…………………………………40
4.4. 根瘤硫酸轉移在E.coli中的表現…………………………41
4.5. 根瘤硫酸轉移的純化…………………………………………45
4.6. N,N'',N''''-Tri-N-acetylchitotriose-6-sulfate的製備47
4.7. N,N'',N''''-Tri-N-acetylchitotriose-6-sulfate結構的鑑.48
4.8. 酵素活性分析方法………………………………………………49
第五章、圖表與說明……………………………………………………54
第六章、參考文獻………………………………………………………75
第七章、附錄……………………………………………………………82
第六章 參考文獻
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