臺灣博碩士論文加值系統

為探討日本血吸蟲穀胱甘呔轉移酵素(Schistosoma japonicum glutathione-S-transferase，SjGST)親和純化系統中，Cys殘基的硫化現象，本論文以基因工程技術，對於C端融合有連續六個His胺基酸之SjGST酵素(簡稱SjGST/His)進行單、雙、三、及四點Cys → Ser定點突變之研究，並分別對於以穀胱甘呔(glutathione，GSH)親和凝膠及固定化金屬親和層析所純化之突變酵素進行活性、酵素動力學、SDS-PAGE、等電點電泳(IEF)、分子量測定、免疫等分析。結果證明以PCR (polymerase chain reaction)方法可成功建構具有Cys169 → Ser (簡稱C169S)突變之SjGST/His基因片段，同時再利用先前已建構並分別具有C85S、C138S、及C178S三個單點突變的SjGST/His基因片段為基礎，進行再重組，可成功得到各種Cys → Ser雙、三、及四突變之SjGST/His突變種。各種突變對SjGST/His之活性及免疫性不會造成嚴重的影響。此外，由SDS-PAGE實驗發現，Cys85殘基會與其它三個Cys殘基間形成分子內雙硫鍵，尤以Cys85和Cys169間之情形較為嚴重。再者，由SDS-PAGE、Maldi-TOF分子量測定及IEF實驗結果發現，四突變之SjGST/His不會造成分子間及分子內雙硫鍵之形成，也不會被SjGST純化系統中之GSH提沖液所修飾，較原生種或其它突變種更適合做為重組蛋白之親和標籤。最後，由動力學研究結果也發現，各種突變對SjGST/His酵素之G-site活性區並不會造成影響，然而對H-site活性區而言，卻會有某些程度的影響，尤以某些含有Cys85突變之SjGST/His有較明顯的變化。總言之，本論文確實建立了一個適用性、穩定性更高的商業化SjGST純化系統，並有助於日本血吸蟲疫苗SjGST/His成份之純化。

The objective of this research was to investigate the thiolation of Cys residues in the Schistosoma japonicum glutathione-S-transferase (SjGST) affinity purification system. A C-terminal 6xHis-tagged SjGST , named as SjGST/His, was studied, as well as its various single, double, triple, and quadruplet Cys → Ser site-directed mutants. Both glutathione (GSH) affinity chromatography and immobilized metal affinity chromatography were used to purify the wild-type and mutant enzymes, followed by the activity, enzymatic kinetics, SDS-PAGE, IEF, molecular weight measurement, and immunoassay studies for all the enzymes. The results showed that the Cys169 → Ser (C169S) SjGST/His mutant was successfully constructed by PCR, and the other various combinations of Cys → Ser mutations were also successfully achieved by recombinant DNA technology. All the mutations did not significantly affect the enzymatic and immunological actives of the SjGST/His enzyme. In addition, the results of SDS-PAGE, Maldi-TOF, and IEF analyses indicated that the neither the intra- nor inter-molecular disulfide bond was formed in the tetra- Cys → Ser SjGST/His mutant, and the mutant was resistant to the GSH thiol-modification. Therefore, the quadruplet mutant was better than the wild type one to be an affinity purification tag for recombinant proteins. Finally, the kinetic studies showed that none of the mutations affected the G-site function of the enzyme, while the Cys85 → Ser mutation had some effects on the H-site function. In conclusion, the new SjGST affinity-tag constructed in this study has provided a more convenient and stable affinity purification system for recombinant proteins in commercial use and a better SjGST component preparation for the vaccine against Schistosomasis.

中文摘要…………………………………………………………………I
英文摘要………………………………………………………………...II
誌謝……………………………………………………………………III
目錄………………………………………………………………..……IV
表目錄……………………………………….…………………………VII
圖目錄…………………………………………………………………..IX
第一章 緒論………………………………………………………….….1
第二章 文獻回顧………….…………………………………………….3
2.1 酵素之特性……………...…………………………………………3
2.2 穀胱甘呔轉移酵素(GST)…………………………………...……..4
2.2.1 GSH ...……………………………………………….……...4
2.2.2 GST………………………………..………………………..5
2.2.3 GST酵素之立體結構……………………..………….…….7
2.2.4 GST上各殘基之研究……………………….…….………11
2.3 重組蛋白質之親和純化…………………….……………………12
2.3.1 固定化金屬親和層析法(IMAC)……...………………….15
2.3.2 SjGST親和層析…………………………………......……22
2.4 蛋白質基因突變法…………………………………………….…23
2.5 蛋白質分析方法………………………………………………….27
2.5.1 GST活性分析……………………………………………..27
2.5.2 等電點電泳（IEF）………………………………………...29
2.5.3 SDS-PAGE蛋白質電泳……………………...………...…30
2.5.4 雷射激發飛行時間質譜儀(MALDI-TOF)…………….…30
第三章 研究目的………………………………………………………32
第四章 研究方法……………………………………………………....34
4.1 實驗流程………………………………………………………….34
4.1.1 pGSTH質體中各種SjGST/His突變基因之再重組…..…34
4.1.2 C169S SjGST/His基因之定點突變…………...………….34
4.1.3 原生種及各突變種SjGST/His蛋白質之生產與純化…...35
4.1.4 原生種及各突變種GST/His酵素之活性與特性分析..…36
4.2 實驗材料…………………………………………………………..42
4.3 其它藥品…………………………………………………………..44
4.4 實驗設備…………………………………………………………..46
4.5 實驗步驟………………………………………...………………..49
4.5.1 SjGST/His基因之突變及再重組…….………...…………49
4.5.2 各種SjGST/His蛋白質之生產與純化…………...………58
4.5.3 各種SjGST/His蛋白質之活性與特性分析…..………….59
第五章 結果與討論……………………….…………………….……..64
5.1 各種SjGST/His突變基因之再重組及C169S突變………….…..64
5.1.1 SjGST/His C138S/C178S雙突變基因之建構與確認…....64
5.1.2 SjGST/His C85S/C138S/C178S三突變基因之建構與
確認…………...……………………………………….….65
5.1.3 SjGST/His C85S/C138S及C85S/C178S雙突變基因之
建構與確認……………………………………………...66
5.1.4 C169S突變基因之確認……………………..…………....66
5.1.5 SjGST/His C85S/C169S雙突變基因之建構與
確認……………………………………………………......67
5.2 各種SjGST/His酵素之生產與純化…………………………….79
5.2.1 GSH Sepharose 4B凝膠純化……………………..………79
5.2.2 Ni-NTA凝膠純化………………………………..………..79
5.3 各種SjGST/His酵素之SDS-PAGE蛋白質電泳分析…..……...87
5.4 Maldi-TOF分子量分析…………………………………………90
5.4.1 以GSH Sepharose 4B凝膠純化之SjGST/His分子量..….90
5.4.2 以Ni-NTA凝膠純化之SjGST/His分子量…………….....90
5.5 各種SjGST/His酵素之IEF電泳分析……………………..………91
5.6 各SjGST/His突變種之動力分析………………………….…..…105
5.7 各SjGST/His突變種之免疫分析……………………………...…105
第六章 結論…………………………………………………………..109
參考文獻………………………………………………………………111
附錄……………………………………………………………….…117

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