臺灣博碩士論文加值系統

太古生物為三域物種中生存範圍最為廣泛且能生存於各種極端環境的生物。當細胞遭受外界環境因子的劇烈變化，如溫度、鹽度、酸鹼等逆境刺激，常會造成胞內新生成蛋白的錯誤摺疊或使已生成的蛋白質結構不穩定，進而失去生理功能及活性並導致聚集蛋白的形成。生物細胞為了維持胞內蛋白正常構形恆定，演化出分子伴護蛋白系統及蛋白酶體系統，以執行胞內蛋白質品質的控管。分子伴護蛋白系統中的DnaK/Hsp70(包含DnaJ及GrpE)系統普遍存在於真核生物與細菌中，也在嗜中溫的太古生物中被發現；其中高鹽甲烷太古生物Methanohalophilus portucalensis FDF1T的分子伴護蛋白dnaK、dnaJ及grpE基因的轉錄表現會受到鹽度及溫度逆境所誘導。本研究將已構築的MpDnaK、MpDnaJ及MpGrpE分別異源表現在E. coli BL21(DE3)RIL並以Ni Sepharose 6 Fast Flow column純化。利用孔雀石綠試劑偵測異源表現的MpDnaK ATP水解酶的活性，獲得催化效率(Catalytic efficiency，Kcat/Km)分別為107.72 (K)、142.28 (K+J)、118.51 (K+E)及235.22 (K+J+E) min-1．mM-1，其中以MpDnaK+J+E的處理組有最高的Vmax 345.10 pmol/min 與低 Km 1.49 mM，顯示添加共伴護蛋白MpDnaJ及MpGrpE會增加MpDnaK對基質ATP結合的親和性。添加共伴護蛋白之MpDnaK+J+E的ATP水解酶的活性於37°C下比活性(24.81 pmole/min/μg )較僅有MpDnaK的比活性(5.64 pmole/min/μg)高出4.39倍。添加化學伴護因子甜菜鹼(0.8 M)有助於提升MpDnaK+J+E的ATP水解酶活性(1.75倍)。以綠螢光蛋白(GFP)及螢火蟲螢光酵素(Luciferase)作為失活蛋白復性實驗的受質，分析MpDnaK系統的對錯誤摺疊蛋白的修復功能，經鹽酸處理(pH 1.5)的綠螢光蛋白及熱處理(40°C)的螢光酵素，MpDnaK+J+E皆能分別協助變性受質蛋白恢復約65.13%及65.46%的活性。此外，添加化學伴護因子10%甘油或0.8 M甜菜鹼於MpDnaK+J+E中，其熱變性的螢光酵素能恢復78.04%與70.86%的活性，由上述結果顯示分子伴護蛋白MpDnaK確實具有ATP水解酶活性及化學伴護因子能提升MpDnaK系統有效地修復失活蛋白的功能及活性。此外重組表現的太古生物型MpDnaK伴護蛋白系統之ATP水解酶最大催化速率明顯高於細菌型EcDnaK伴護蛋白系統約19倍；且在修復螢光酵素活性上，MpDnaK伴護蛋白系統亦優於EcDnaK伴護蛋白系統，顯示太古生物型MpDnaK分子伴護系統具應用於生物醫學相關去聚集蛋白的潛力。

Molecular chaperone plays an important role to assist newly synthesized polypeptides folding, and protects cells from stress induced proteins misfolding and aggregation. Heat shock protein DnaK belongs to molecular chaperone and cooperates with co-chaperone DnaJ and nucleotide exchange factor GrpE. And ATP is required for DnaK to activate and remodel their substrate client proteins. The molecular chaperone system DnaK (Hsp70) system is highly conserved in sequence and distribution in Bacteria and Eukarya, however, DnaK is also found in mesophilic archaeon. The dnaK, dnaJ and grpE genes from halophilic methanoarchaea Methanohalophilus portucalensis FDF1T have been demonstrated that could be induced by the salt and heat stresses. In this study, the dnaK, dnaJ and grpE gene from Methanohalophilus portucalensis FDF1T were heterologously overexpressed in E. coli BL21(DE3)RIL and the His6-tagged proteins were purified by Ni SepharoseTM 6 Fast Flow resin column. The ATPaes activities of MpDnaK system were measured by malachite green phosphate assay. The ATPase catalytic efficiency (Kcat/Km) of MpDnaK alone, K+J, K+E, and KJE were 107.72, 142.28, 118.51, and 235.22 min-1, respectively. The ATP binding kinetic of MpDnaK+J+E reached the highest value with Vmax at 345.10±11.90 pmol/min and low Km at 1.49±0.25 mM. These results indicated that ATPase activity of MpDnaK was stimulated by the addition of co-chaperone MpDnaJ and MpGrpE. The recombinant MpDnaK, MpDnaJ and MpGrpE are stable at 37°C, and the specific ATPase activity of MpDnaK alone (5.64 pmole/min/μg) were increased to 4.39 fold with co-chaperone MpDnaJ and MpGrpE (24.81 pmole/min/μg). Moreover, the ATPase activity of MpDnaK+J+E was increasd 1.75 fold with the additional of 0.8 M chemical chaperone betaine. The green fluorescent protein (GFP) and Luciferase were used as substrates for refolding activity. The the acid-denatured GFP was 65.13% recovered by MpDnaK+J+E. And the presence of MpDnaK+J+E could rescure 65.46% heat (40°C) denatured Luciferase. MpDnaK+J+E with the addition of chemical chaperone glycerol or betaine shows that 78.04% and 70.86% native Luciferase activity was recovered. Our results demonstrated that MpDnaK chaperone system have both ATPase and refolding activities. Moreover, the archaeal MpDnaK system showed higher ATPase and refolding activities than bacterial EcDnaK system which indicated the promising application potential in aggregated protein control.

中文摘要...........................I
英文摘要...........................II
目錄...........................III
表目錄...........................V
圖目錄...........................VI
壹、前言...........................1
貳、前人研究...........................3
一、細胞內聚集蛋白的形成...........................3
二、分子伴護蛋白 (Molecular chaperones)...........................4
(一)ClpB/Hsp100...........................5
(二)DnaK 分子伴護蛋白系統...........................6
(三)分子伴護因子(Chaperonins)...........................8
三、蛋白酶體系統...........................10
四、太古生物的分子伴護蛋白...........................13
五、高鹽甲烷太古生物Methanohalophilus portucalensis FDF1T中的分子伴護蛋白DnaK/DnaJ/GrpE...........................14
參、材料與方法...........................17
一、菌種...........................17
二、大腸桿菌培養基製備、培養與保存...........................17
(一)培養基製備...........................17
(二)大腸桿菌培養與保存...........................17
三、聚合酶連鎖反應(PCR) ...........................18
四、核酸膠體電泳分析及記錄...........................18
五、DNA片段純化與回收...........................19
六、DNA黏合反應...........................19
(一)pGEMR-T Vector System...........................19
(二)其他非商業化的質體...........................20
七、勝任細胞製備與轉型作用...........................20
(一)勝任細胞製備...........................20
(二)轉型作用...........................20
八、質體抽取及純化...........................21
九、構築Luciferase蛋白表現載體與表現系統...........................21
十、蛋白質表現與純化...........................22
(一)誘導表現MpDnaK、MpDnaJ及MpGrpE蛋白...........................23
(二)誘導表現綠螢光蛋白 (Green flourecent protein, GFP)...........................23
(三)誘導表現螢光酵素 (Luciferase) 蛋白...........................24
(四)蛋白質定量...........................24
(五)親和性交換樹脂Ni SepharoseTM 6 Fast Flow resin column純化蛋白...........................24
1.純化MpDnaK、MpDnaJ及MpGrpE蛋白...........................25
2.純化綠螢光蛋白 (GFP) 及螢光酵素 (Luciferase) ...........................26
(六)蛋白質濃縮...........................26
十一、蛋白質電泳 (SDS-PAGE)...........................27
十二、MpDnaK伴護蛋白系統之ATP水解酶活性測試...........................28
十三、受質蛋白復性實驗...........................29
(一)化學變性-綠螢光蛋白 (GFP)...........................29
(二)物理變性-螢光酵素 (Luciferase, Luc)...........................30
肆、結果與討論...........................32
一、MpDnaK、MpDnaJ及MpGrpE蛋白表現及純化...........................32
二、受質蛋白綠螢光蛋白 (GFP) 的蛋白表現與純化...........................33
三、螢光酵素 (Luciferase, Luc) 表現系統構築、Luciferase蛋白表現與純化...........................33
四、MpDnaK伴護蛋白系統之ATP水解酶活性測試...........................34
(一)MpDnaK ATP水解酶活性與反應時間的關係...........................34
(二)MpDnaK ATP水解酶活性與ATP基質濃度的關係...........................35
(三)溫度對MpDnaK伴護蛋白系統ATP水解酶活性之影響...........................37
(四)甜菜鹼對MpDnaK伴護蛋白系統ATP水解酶活性之影響...........................37
(五)鉀離子對MpDnaK伴護蛋白系統ATP水解酶活性之影響...........................38
五、MpDnaK伴護蛋白系統之受質復性分析...........................39
(一)MpDnaK伴護蛋白系統對酸變性綠螢光蛋白 (GFP) 之復性結果...........................39
(二)MpDnaK伴護蛋白系統對熱變性螢光酵素 (Luciferase) 蛋白之復性結果...........................40
(三)化學伴護因子與MpDnaK伴護蛋白系統修復熱變性的螢光酵素(Luciferase) 之影響........................... 41
伍、結論與展望...........................43
陸、表與圖...........................45
柒、參考文獻...........................62

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