Optimized Expression and Purification of Recombinant Kir6.2 delta C26 Channel Protein

腺核苷三磷酸感受性鉀離子通道 (KATP channels) 由兩種蛋白組成一異八分子聚合體：其中第六型內向性整流型鉀離子通道蛋白 (Kir6.x) 形成鉀離子篩選孔洞；而磺脲受體蛋白 (SUR) 則與調控有關。此通道廣泛分佈於不同組織：包括胰臟β細胞、心肌與骨骼肌細胞，並受細胞內之腺核苷三磷酸 (ATP)、二磷酸脂酰肌醇 (PIP2) 及MgATP濃度所調控。第六型內向性整流型鉀離子通道蛋白 (Kir6.2) 構造上主要有兩個過膜區域；生理功能主要是負責運送鉀離子及穩定靜止膜電位，與神經系統保護作用有關；若Kir6.2發生突變，則會導致永久性新生兒糖尿的發生。直至目前為止，研究者尚未成功獲致Kir6.2蛋白結晶。因此，有關核苷酸如何調控離子通道活性及開關的細部機制至今仍不清楚。本篇論文主要希望能夠最佳化表現及純化Kir6.2融合蛋白，以進一步獲致其蛋白質單晶晶體結構。 我們利用不同E. coli表現菌株，將C端26個胺基酸序列去除掉的Kir6.2蛋白基因 (Kir6.2 delta C26) 接入pET20b(+)及pET28a(+)表現載體中，使其分別及同時在Kir6.2的N、C端都帶有6xHis-Tag以減少對表現蛋白的結構影響，另外亦接入pGEX4T-1中，建構一帶有GST-tag之融合蛋白以增加蛋白之溶解性。前結果顯示目標蛋白均無法成功的被誘導表現，甚至對於E. coli宿主細胞有毒殺作用。雖然醣化作用對於Kir6.2機能上非絕對必須，但是考慮到腺核苷三磷酸感受性鉀離子通道是個需要高度調控的分子，我們亦嘗試利用較高等的表現系統，如：酵母菌及昆蟲細胞。預期能夠因適當的調控而減少對表現細胞的傷害及增加蛋白的產量。結果顯示只純化出少量蛋白質，此外，我們將試著進行重複基因插入並增加昆蟲細胞之Transfection Efficiency以獲得高產量的純化蛋白。ATP sensitive potassium (KATP) channels are hetero-octamer of two proteins: The K+-selective channel pore is forms with Kir6.x (inwardly rectifying potassium channels) and the regulatory subunit is composed of SUR (sulphonylurea receptors). They are distributed in various tissues include the pancreatic β-cell, cardiac and skeletal muscle are affected by intracellular ATP, PIP2 and MgATP. The topological and physiological characteristics of Kir6.2 are two transmembrane domains and transfer the K+ across the membrane and stabilizing the resting membrane potential. It related to the neuroprotection and loss-of-function in Kir6.2 cause permanent neonatal diabetes. Crystallization of Kir6.2 has not proved successful. The detail mechanism of the nucleotides regulating the channel activity and gating have not been defined so far. We attempted to express and purify the fusion proteins of Kir6.2 to determine the single crystal structure. Kir6.2 delta C26 was in-frame subcloned into pET20b, pET28a with hexhistidine tag in its N and/or C terminus to reduce the effect of the co-expressed protein and pGEX4T-1 vector with GST-tag to increase the solubility of fusion protein in various E. coli strains. ntil now, it was found from the results that the target protein has not induced successful and maybe toxic to the E. coli host cell. Although glycosylation is not prerequisite for functional Kir6.2. In order to consider ATP sensitive potassium (KATP) channels which need highly regulation, we also try performance advanced expression system as: yeast and insect cell. Expression with yeast and inset cell is expected to decrease toxicity and increase the production. The data come out less protein was purified. Furthermore, we will try the gene replica insertion events and raise the transfection efficiency of the Baculovirus-insect cells system to obtain the pure protein in large-scale.口試委員會審定書…………………………………………………...……1文摘要……………………………………………………………...……2bstract (英文摘要)…………………………………………………...….. 3寫檢索表…………………………………………………………...…....8一章緒論…………………………………………………………...…..10.1 鉀離子通道…………………………………………………...…10.2 內向性整流型鉀離子通道 ………………………………….…11.3 腺核苷三磷酸感受性鉀離子通道的調控…………………...…12.4 內向性整流型鉀離子通道Kir6.2之臨床意義……………...….14.5 研究動機……………………………………………………...…14二章 實驗材料與方法……………………………………………...…16.1 Kir6.2核苷酸序列………………………………...……………..16.2 蛋白表現載體……………………………………...……………16.3 菌株及細胞………………………………………...……………16.4 藥品………………………………………………...……………16.5 實驗儀器及電腦軟體……………………………...……………19.6 實驗方法…………………………………………...……………20.6.1 Kir6.2重組基因萃取…………………………...…………...20.6.2 聚合酶連鎖反應 (Polymerase Chain Reaction; PCR)….....21.6.3 瓊脂凝膠 (Agarose Gel) 之DNA回收……………..…….22.6.4 接合反應 (Ligation)……………………………………......23.6.5 勝任細胞 (Competent cell) 的製備…………………….....23.6.6 轉型反應 (Transformation)……………………………...…23.6.7 E. coli (DH5α) 菌落聚合酶連鎖反應 (Colony PCR)…......24.6.8 蛋白表現系統之製備……………………………………....25 2.6.8.1 E. coli表現菌株的製備…………………………...…..25 2.6.8.2 Spheroplasting of P. pastoris GS115的製備……...…..25 2.6.8.2.1 P. pastoris GS115之轉型反應………………...….26 2.6.8.2.2 P. pastoris菌落聚合酶連鎖反應……………...….27 2.6.8.3 Baculovirus-Insect cell表現系統…………………..…27.6.9 大腸桿菌系統之標的蛋白表現………………………...….27.6.10 酵母菌系統之標的蛋白表現…………………………......28.6.11 以His-tag親和性管柱純化Kir6.2融合蛋白質………......29.6.12 西方墨點法 (Western Blotting)………………………..…30.6.13 桿狀病毒-昆蟲細胞系統之標的蛋白表現…………….....33.6.14 桿狀病毒-昆蟲細胞系統表現蛋白之純化…………….....34三章 實驗結果與討論………………………………………………...36四章 結論…………………………………………………………...…42五章 參考文獻……………………………………………………...…44六章 實驗圖表………………………………………….……….…….4