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研究生:曾麗容
研究生(外文):Li-Jung Tseng
論文名稱:Kir1.1鉀離子通道胞內區域之表現,純化及三度空間結構的模擬
論文名稱(外文):Expression, purification, and 3-D homology modeling of intracellular domains of Kir1.1 channel
指導教授:樓國隆劉宏輝劉宏輝引用關係
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:口腔生物科學研究所
學門:醫藥衛生學門
學類:牙醫學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
中文關鍵詞:內向性整流型鉀離子通道電腦模擬蛋白質嵌合
外文關鍵詞:inwardly rectifying potassium channelcomputer modelingprotein docking
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腎臟外髓質上的內向性整流型鉀離子通道(Renal outer medullary potassium channel; ROMK; Kir1.x)是屬於Kir鉀離子通道的第一族,廣泛的分佈於體內而且調控著許多重要的生理功能,包括:維持細胞靜止膜電位、神經突觸之興奮性,和腎臟鉀離子的運輸。已知ROMK1鉀離子通道之磷酸與去磷酸化的過程調控著離子通道的活化,而近年來的研究指出ROMK1鉀離子通道之磷酸化的調控是透過蛋白質激酶A (cyclic AMP-dependent protein kinase)的作用且藉由PIP2的參與而產生的。另外ROMK1鉀離子通道也被證明會受到細胞內酸鹼值(pHi)影響;當細胞內酸鹼值下降時,會引起ROMK1鉀離子通道的關閉。目前推測這些造成ROMK1鉀離子通道的開閉情形可能與通道膜內區域構形的重組有關,然而其細部的機制卻不清楚,因此我們計劃進行膜內區域的晶體結構測定。由於晶體結構測定需要較長的研究進行,所以本論文中,我們企圖以蛋白質表現、純化的前期作業和分子模擬及模型建立來作為短期目標。
本研究中我們建構了ROMK1鉀離子通道之N端與C端的GST融合蛋白質並試著去大量表現與純化,初步已得到C端的融合蛋白質,目前正朝著表現N端的融合蛋白質以及提高C端的質與量邁進。而藉由電腦模擬與蛋白質嵌合,對於一些調控的機制已能夠有相當程度之結構功能的詮釋。
Renal outer medullary potassium channels (ROMK; Kir1.x) belong to the first family of Kir potassium channels and are widely distributed in various tissues. They regulate many important physiological functions, including the maintenance of resting potential and of neuron synaptic excitability, as well as the renal K+ transport. Recently, the phosphorylation and dephosphorylation involved in the activation of ROMK1 channels have been suggested to be mediated by cyclic AMP-dependent protein kinase through the participation of PIP2 molecule. In addition, it is generally believed that the ROMK1 channel activity can be strongly influenced by the intracellular pH (pHi). Decrease in pHi will bring the ROMK1 channels towards their closed state. Such induced channel closure may be associated with the structural rearrangement of the intracellular domains with which, however, no detailed mechanism is so far yet available. Therefore, we would like to, as a long-term goal, determine the crystal structure of the intracellular domains of ROMK1 channels with the expression, purification and 3-D modeling as frontier studies presented in the thesis.
We have constructed the GST-fusion proteins of N- and C- termini of ROMK1 with the C-terminal part successfully expressed and purified. Furthermore, the results based on computer modeling and docking simulation are hopefully to provide fresh insights into the structural-functional correlation explaining the intracellular events of pH effects and PIP2 interaction.
目錄----------------------------------------------------------------------------------------1
圖表目錄------------------------------------------------------------------------------------------4
附錄目錄------------------------------------------------------------------------------------------5
英文縮寫表---------------------------------------------------------------------------------------6
英文摘要------------------------------------------------------------------------------------------7
中文摘要------------------------------------------------------------------------------------------9
前言--------------------------------------------------------------------------------------10
第一節 內向性整流型鉀離子通道---------------------------------------------------------10
第二節 腎臟外髓質上的內向性整流型鉀離子通道------------------------------------13
第三節 ROMK1鉀離子通道的調控機制-------------------------------------------------15
第四節 蛋白質交互作用的預測------------------------------------------------------------17
第五節 研究動機與目的---------------------------------------------------------------------18
實驗材料--------------------------------------------------------------------------------19
一、 菌株---------------------------------------------------------------------------------------19
二、 載體---------------------------------------------------------------------------------------19
三、 試劑與藥品配方------------------------------------------------------------------------19
1. 試劑-------------------------------------------------------------------------------------19
2. 藥品配方-------------------------------------------------------------------------------21
四、 實驗儀器---------------------------------------------------------------------------------26
五、 電腦硬體---------------------------------------------------------------------------------27
六、 電腦軟體---------------------------------------------------------------------------------27
實驗方法--------------------------------------------------------------------------------28
一、 質體之建構-------------------------------------------------------------------------------28
1. 載體的消化---------------------------------------------------------------------------28
2. 連接載體與插入片段---------------------------------------------------------------28
二、 將重組質體轉形至E. coli中-----------------------------------------------------------29
1. 製作適能大腸桿菌------------------------------------------------------------------29
2. 轉形反應------------------------------------------------------------------------------29
3. 質體DNA之小量製備--------------------------------------------------------------30
4. 篩選重組質體------------------------------------------------------------------------30
5. 聚合酶連鎖反應--------------------------------------------------------------31
I. PCR使用之引子序列-----------------------------------------------------------------31
II. PCR的溫度循環條件----------------------------------------------------------------31
III. PCR的混合液-----------------------------------------------------------------------32
三、 大腸桿菌系統之蛋白質的表現與純化----------------------------------------------32
1. 確立蛋白質表現時間及誘導濃度------------------------------------------------32
2. 利用pGEX4T-1衍生質體大量表現蛋白質-------------------------------------33
3. GST親和性管柱純化蛋白質--------------------------------------------------34
四、ROMK1的N端與C端之3-D結構模型的建立------------------------------------34
1. 模板的搜尋--------------------------------------------------------------------------35
2. 模型的建立--------------------------------------------------------------------------35
五、利用Z-Dock分析蛋白質與蛋白質之間的作用--------------------------------------35
實驗結果--------------------------------------------------------------------------------37
一、 ROMK1胞內區域之C端或N端的質體建構----------------------------------------------------------------------------------------------37
1. 質體之確認-限制酶圖譜-----------------------------------------------------------37
二、 ROMK1胞內區域之融合蛋白的表現-----------------------------------------------37
1. 融合蛋白質之表現時間及誘導濃度的分析------------------------------------------------------------------------------------------37
三、 融合蛋白的純化-------------------------------------------------------------------------38
1. ROMK1胞內區域之C端融合蛋白的純化------------------------------------------------------------------------------------------38
四、 ROMK1之N端與C端的3-D結構模型的建立------------------------------------39
1. BLAST系統與KEGG系統搜索的結果-----------------------------------------39
2. 序列排列的結果---------------------------------------------------------------------40
五、 模擬ROMK1之N端與C端的蛋白質與蛋白質之交互作用--------------------40
討論--------------------------------------------------------------------------------------42
一、表現融合蛋白質的方面------------------------------------------------------------------42
二、純化C端之融合蛋白質的方面---------------------------------------------------------44
三、電腦模擬的方面---------------------------------------------------------------------------44
四、蛋白質嵌合的方面------------------------------------------------------------------------45
五、未來的方向---------------------------------------------------------------------------------46
參考文獻--------------------------------------------------------------------------------47
圖表-----------------------------------------------------------------------------------------
附圖-----------------------------------------------------------------------------------------
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