Abstract
AbstractProtein-DNA complexes play a important role in various biological processes transcription, translation and DNA replication. In order to understand this higher order molecular assembly we have taken a SRY related HMG Box (SOX9) protein from zebra fish as a model protein. The theoretical model of human SOX9 was used as the molecular model for the understanding of molecular level interactions.The DNA binding affinity of zfSOX9 was tested using EMSA (Electrophoretic mobility shift assay) analysis with two different DNA probes,S9WT (5-TAAGAACAATGGGA-3) and COL2C1 (5-CCCACAATGCC-3), that found to bind with SOX9 HMG domain.Among the various mutants of zfSOX9 HMG domain, W43A and F12A were found to show high stability against thermal and chemical denaturation, so W43A and F12A were reduced the DNA binding affinity. Modification of F12 (phenylalanine at position twelve) to tyrosine greatly were reduced the DNA binding affinity. Other mutants that show interesting results were N10A and M13A, even though these mutants showed stability comparable to that of wild type protein, the DNA binding ability was very poor. Alanine site directed mutant analysis of the residues at positions M9, S36, K37, K66, K73 and Y74 did not alter the stability or the DNA binding affinity. Modification of highly conserve residue of HMG domain protein family H65 and P70 did not show any remarkable change in the SOX9 HMG box protein biophysical property.Abstract……………………………………………………………………..…..1中文摘要……………………………………………………………………….2第一章 緒論第一節 HMG 的特性介紹……………………………………….....3第二節 Sox9的功能介紹……………………………………….…..5第三節 SOX9-HMG的結構和功能介紹………………....……..8第四節 實驗目的……………………………………………….….…8圖1 SRY HMG domain 與DNA之複合體………….………...10圖2 zfSOX9 HMG結構區的氨基酸序列比對……..………….11圖3 HMG(SOX9)與HMG(SRY)的氨基酸序列比對………...12第二章 材料與方法第一節 SOX9HMG結構區的載體…………………………….14第二節 SOX9HMG結構區的DNA突變設計與取得……..14一、 突變引子(primer)的設計…………………………………...15表 1氨基酸殘基取代成丙安酸(Alanine) ………….………...15二、 突變引子(primer)的合成…………………………………...16三、 聚合脢連鎖反應……………………………………………...16四、 DNA洋菜膠電泳分析圖(DNA agarose gel)……………17五、 將突變DNA轉殖於XL1-blue component cell………..181. XL1-blue component cell的備製……………………..…18六、 載體萃取與DNA定序……………………………………..19第三節 Sox9 HMG結構區蛋白質的備製……………………..19一、 將成功突變的質體DNA轉殖到BL21 DE3 勝任細胞二、 蛋白質的表現與純化……………………………………….211. 蛋白質表現的小量測試…………………………….….…212. 蛋白質的大量表現與萃取…………………………..……213. 蛋白質的純化……………………………………………….22第四節 蛋白質的分析………………………………………………23一、 蛋白質濃度測定……………………………………………...23二、 蛋白質質量的測定(Mass) …………………………………24三、 EMSA(Electrophoretic mobility shift assay) ……………24四、 旋光儀光譜分析………………………………………………251. 旋光儀光譜圖(Wavelength scan) ………………………..272. 熱變性實驗(Thermal Denature Experiments) …………273. 化學變性實驗(Chemical denaturation experiments)…28圖4. Sox9HMG結構區的載體（vector）……………………..30圖5. 旋光儀光譜圖………………………………………………..31第三章、實驗結果與討論第一節、 SOX9 -HMG結構區的DNA突變取得…………….32一、 DNA洋菜膠體電泳分析…第二節、 SOX9 -HMG結構區蛋白質的備製………………….32一、 蛋白質表達之測試：小量測試…………………………..32二、 蛋白質的純化……………………………………………...…32三、 Thrombin之測試…………………………………………….33四、 HPLC純化SOX9 HMG蛋白質………………………….33第三節、 蛋白質的分析…………………………………………….33一、 蛋白質分子量質譜儀分析…………………………………33二、 EMSA(Electrophoretic mobility shift assay)……………34三、 旋光儀光譜分析……………………………………………..36第四章 實驗結果圖表………………………………………………...41第一節、 SOX9 -HMG結構區的DNA突變取得…………….41一、 DNA洋菜膠體電泳分析......................................................41圖6 .1﹪DNA洋菜膠分析聚合脢連鎖反應產物………….41第二節、 SOX9 -HMG結構區蛋白質的備製…………………42一、 蛋白質表達之測試：小量測試………………………42圖7. 15﹪SDS-PAGE分析原型SOX9 HMG和突變SOX9 HMG的表現量。..................................................................42二、 蛋白質的純化………………………………………………...43圖8. 16.5﹪SDS-PAGE分析原型SOX9 HMG結構區經Ni2+-column純化……………………………………………………43三、 Thrombin之測試……………………………………………..44圖9. 16.5﹪SDS-PAGE分析原型SOX9 HMG加入Thrombin的時間測試圖..................................................................44圖9-1純化蛋白質序列...................................................................45圖9-2 原型SOX 9 HMG結構區的HPLC純化圖……………46第三節、 蛋白質的分析…………………………………………….47一、 蛋白質分子量質譜儀(mass)分析……………………..47圖 10.SOX9 M9A質譜儀分析圖..................................................47圖 11. SOX9 N10A質譜儀分析圖……………………………..47圖 12.SOX9 M13A質譜儀分析圖................................................48圖 13.SOX9 H29A 質譜儀分析圖...............................................48圖 14.SOX9 S36A 質譜儀分析圖...............................................49圖 15.SOX9 K37A質譜儀分析圖................................................49圖 16.SOX9 W43A 質譜儀分析圖..............................................50圖 17.SOX9 H65A質譜儀分析圖................................................50圖 18.SOX9 P70A質譜儀分析圖..................................................51圖 19.SOX9 K73A質譜儀分析圖................................................51圖 20.SOX9 Y74A質譜儀分析圖................................................52二、 EMSA(Electrophoretic mobility shift assay…………….....53圖21. 20% nondenaturing PAGE gel for原型SOX9對S9WT、COL2C1的結合測試……………………………………………….54圖22. 20% nondenaturing PAGE gel for原型SOX9結構區和突變SOX9結構區對S9WT、COL2C1的結合測試。……….55圖23.20% nondenaturing PAGE gel for原型SOX9結構區和突變SOX9結構區對COL2C1的結合測試………………………….56圖 24.20% nondenaturing PAGE for 原型SOX9結構區和突變SOX9結構區對COL2C1的結合測試………………………….57圖 25.20% nondenaturing PAGE for 原型SOX9結構區和突變SOX9結構區對S9WT的結合測試………………………………58圖 26. 20% nondenaturing PAGE gel for原型SOX9結構區和突變SOX9結構區對S9WT的結合測試…………………………59圖 27.20% nondenaturing PAGE gel for 原型SOX9結構區和突變SOX9結構區對S9WT的結合測試…………………………60圖 28.20% nondenaturing PAGE gel for 原型SOX9結構區和突變SOX9結構區對S9WT的結合測試…………………………61三、 旋光儀光譜分析……………………………………………..621.原型ＳOX9 HMG結構區和突變ＳOX9 HMG結構區的螺旋性比較………………………………………………………………….62A.螺旋性實驗……………………………………………………....62圖 29.原型SOX9 HMG結構區的旋光儀光譜圖……………62圖30.原型SOX9和M9A、N10A、M13A、H29A的旋光儀光譜圖……………………………………………………………………..63圖31.原型SOX9和S36A、K37A、W43A的旋光儀光譜圖……………………………………………………………………………64圖32.原型SOX9和H65A、K66A、P70A、K73A、Y74A的旋光儀光譜圖…………………………………………………………….65B熱變性實驗 (Thermal Denature Experiments)…………….66圖33.原型SOX9的熱變性旋光儀光譜圖…………………….66圖35.原型SOX9-HMG結構區和M9A、N10A、M13A和H29A的熱變性旋光儀光譜圖……………………………………………..68圖37.原型SOX9 HMG和S36A、K37A、W43A的熱變性旋光儀光譜圖……………………………………………………………….70圖39.原型SOX9 HMG和K66A、P70A、K73A和Y74A的熱變性旋光儀光譜圖…………………………………………………..72C. 化學變性實驗Chemical denaturation experiments)…………………………………………………………………………75圖42.原型SOX9 HMG之化學變性實驗……………………...75圖43.原型SOX9 HMG和M9A、N10A之化學變性實驗..76圖45.原型SOX9 HMG和S36A、K37A、E50A之化學變性實驗…………………………………………………………………………78圖47.原型SOX9、P70A、K73A和Y74A之化學變性實驗……………………………………………………………………………80圖 49.突變SOX9 HMG結構區-DNA 複合體的相對位置..82圖 50.突變SOX9 HMG結構區-DNA 複合體的相對位置..83圖 51.突變SOX9 HMG結構區-DNA 複合體的相對位置..84圖 52.突變SOX9 HMG結構區-DNA 複合體的相對位置..85圖 53.突變SOX9 HMG結構區-DNA 複合體的相對位置..86表 2.EMSA表為原型SOX9 HMG和突變SOX9 HMG結合DNA後的統整表……………………………………………………..87表3. 旋光儀對各突變蛋白質特性分析與原型蛋白質之對照表(含螺旋性、熱穩定性及化學變性的統整)…………………….88表4.各突變蛋白質特性分析與原型蛋白質之對照表(實驗室資料)………………………………………………………………………..89表5. 各突變蛋白質特性分析與原型蛋白質之對照表(包含螺旋性、Tm、Cm、EMSA統整)………………………………………90表6.hSRY HMG和LEF HMG與各別探針DNA的結合關係…………………………………………………………………………….91第五章 參考資料…………………………………………………………92