樓國隆臺灣大學：口腔生物科學研究所許碧絨Hsu, Pi-JungPi-JungHsu2007-11-262018-07-092007-11-262018-07-092005http://ntur.lib.ntu.edu.tw//handle/246246/51310內向性整流型鉀離子通道Kir6.2 (Inwardly rectifying potassium channels; Kir6.2)屬於Kir離子通道中的第六族，廣泛分布於身體不同的組織，特別在腦部、骨骼肌和胰臟β細胞中最多，並且調控許多重要生理功能如維持細胞靜止膜電位、胰島素分泌以及骨骼肌的收縮等。近十年的研究已知SUR (sulphonylurea receptor)分子與調控Kir6.2的開啟有關，根據電生理的研究分析而知 ATP、MgADP、PIP2 (phosphatidyl-inositol-4,5 bisphosphate)也與調節Kir6.2有關，但無法得知分子間細部的作用機制，因此計劃進行蛋白質晶體結構測定。由於晶體結構測定需要較長的研究進行，所以本論文的目的在於表現與純化出Kir6.2離子通道的蛋白質，以作為進行蛋白質晶體結構測定之前期作業。 目前我們建構了Kir6.2離子通道與His-tag之融合蛋白，並表現於大腸桿菌系統中。根據電泳與西方點墨法結果顯示其蛋白已成功表現並得正確分子量。有趣的是施以高濃度的尿素無法將pellet的蛋白萃取出來，這種獨特的現象可能表示蛋白嚴重錯誤的疊合，因此進一步的純化以及使蛋白再正確疊合的處理為未來的首要目標。Inwardly rectifying potassium channels Kir6.2 belong to the sixth family of Kir potassium channels. They are widely distributed in various tissues, especially in brain, skeletal muscles, and pancreatic β-cells. They regulate many important physiological functions, including the maintenance of resting potentials, the secretion of insulin and the contraction of skeletal muscles. The sulphonylurea receptor (SUR) subunit has been reported since a decade involving the regulation of the opening of Kir6.2 channels. In addition, such gating behavior was also suggested to be mediated by ATP, MgADP, and PIP2 based upon the electrophysiological analysis. The detailed mechanism remains to be identified. Therefore, we would like to, as a long-term goal, determine the crystal structure of the Kir6.2 channels with the expression and purification as pioneering studies presented in the thesis. We have constructed the His-tag fusion protein of Kir6.2 channels and expressed with E. coli system. Results from electrophoresis and western blotting suggested successful expression of desired proteins in correct molecular weight. Interestingly, treatment of high concentration urea did not extract our protein from the pellets. Such unique phenomenon may imply a severe misfolding problem. Therefore, further purification and refolding treatment should the next leading concern in the near future.縮寫表-------------------------------------------VI 中文摘要-----------------------------------------1 英文摘要-----------------------------------------2 第一章 前言------------------------------------3 1. 1內向性整流型鉀離子通道-----------------------3 1.1.1內向性整流型鉀離子通道的基本特性------------3 1.1.2內向性整流型鉀離子通道的生理角色------------4 1.1.3內向性整流型鉀離子通道家族------------------5 1.1.4內向性整流型鉀離子通道的構形----------------6 1.2內向性整流型鉀離子通道Kir6.2------------------7 1.2.1 Kir6.2離子通道的特性-----------------------7 1.2.2 Kir6.2離子通道的分布-------------------8 1.2.3 Kir6.2離子通道的調控機制---------------9 1.2.4與Kir6.2離子通道相關之疾病--------------11 1.3研究動機--------------------------------------13 第二章 實驗材料與方法----------------------------14 2.1實驗材料--------------------------------------14 2.1.1 Kir6.2 DNA sequence-------------------14 2.1.2 菌株----------------------------------14 2.1.3 載體----------------------------------14 2.1.4 試劑----------------------------------14 2.1.5 藥品配方------------------------------18 2.1.5.1 培養基與培養液-----------------18 2.1.5.2 緩衝溶液配方-------------------19 2.1.5.3 凝膠配方-----------------------23 2.1.6實驗儀器及電腦軟體---------------------24 2.2 實驗方法-------------------------------------26 2.2.1質體之建構----------------------------------27 2.2.1.1重組質體Kir6.2/ pET21a+的抽取與切割--27 2.2.1.2瓊脂凝膠之Kir6.2 DNA回收-------------28 2.2.1.3 載體pET20b+的切割-------------------29 2.2.1.4 連接載體pET20b+與Kir6.2DNA----------29 2.2.2重組質體轉型至大腸桿菌系統------------------29 2.2.2.1 適能大腸桿菌來源--------------------29 2.2.2.2 轉形反應----------------------------30 2.2.2.3 重組質體Kir6.2/ pET20b+之小量製備---30 2.2.2.4 篩選重組質體Kir6.2/ pET20b+---------31 2.2.2.5 聚合酶連鎖反應----------------------31 2.2.3大腸桿菌系統之蛋白質的表現------------------33 2.2.3.1確立Kir6.2蛋白質表現時間及誘導濃度---33 2.2.3.2 利用pET20b+衍生質體大量表現Kir6.2蛋白質-34 2.2.3.3西方點墨法確認Kir6.2蛋白質-----------34 2.2.3.4 利用尿素萃取Kir6.2融合蛋白質形成的包涵體-36 2.2.4 以His-tag親和性管柱純化Kir6.2融合蛋白質----36 第三章 實驗結果---------------------------------38 3.1 重組質體Kir6.2 / pET20b+之確認與限制酶圖譜---38 3.2 Kir6.2 / pET20b+轉形至大腸桿菌篩選結果-------38 3.3 Kir6.2融合蛋白質之表現及誘導濃度的分析-------39 3.4利用尿素萃取出Kir6.2融合蛋白質形成的包涵體之結果-39 3.5以His-tag親和性管柱純化Kir6.2融合蛋白質結果---40 3.6以His-tag親和性管柱純化Kir6.2融合蛋白質結果---40 第四章 討論----------------------------------41 4.1 重組質體Kir6.2 / pET20b+之確認---------------41 4.2 Kir6.2融合蛋白質之誘導濃度分析---------------41 4.3 Kir6.2融合蛋白質之表現分析-------------------42 4.4 利用尿素萃取出Kir6.2融合蛋白質形成的包涵體---43 4.5以His-tag親和性管柱純化Kir6.2融合蛋白質-------43 第五章 結論與未來方向------------------------45 參考文獻-----------------------------------------46 實驗圖表-----------------------------------------511013657 bytesapplication/pdfen-US融合蛋白內向性整流型鉀離子通道Inwardly rectifying potassium channelsfusion proteinotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/51310/1/ntu-94-R91450015-1.pdf