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標題: 高鹽甲烷太古生物Methanohalophilus portucalensis FDF1T鉀離子通道蛋白 基因選殖、異源表現與特性分析
Cloning, Expression, and Functional Characterization of Potassium Channel from Methanohalophilus portucalensis FDF1T
作者: 王俊文
Wang, Jiun-Wen
關鍵字: 太古生物;Archaea;高鹽甲烷菌;鉀離子通道;膜電壓感應器;Halophilic methanogen;Potassium channel;Voltage sensor
出版社: 生命科學系所
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鉀離子是細胞內最主要的陽離子,並且在滲透壓調控以及酸鹼平衡中扮演著重要的角色。鉀離子通道可快速運送鉀離子,每秒達107個鉀離子。生長在高達2.9 M鹽濃度環境的高鹽甲烷太古生物Methanohalophilus portucalensis FDF1T可在細胞內累積1.13 M濃度的鉀離子以及低分子量的有機分子作為相容質抵抗外界鹽濃度壓力。本研究利用M. portucalensis FDF1T基因解序所得到的序列獲得完整的鉀離子通道基因(kvmp)全長。TOPCONS分析KvMp二級結構具有六個穿膜片段,多重胺基酸序列比對顯示KvMp具有典型的Voltage sensor(RXXRXXRXXK) 以及selectivity filter(TXGYGD)之保守序列。將高鹽太古生物的kvmp轉殖到鉀離子運輸缺陷的E. coli LB2003,表現KvMp的轉殖株可生長於在低鉀離子KML(添加0, 1 ,10 mM) 培養基,顯示KvMp蛋白具有鉀離子通道的功能。證實甲烷太古生物的鉀離子通道可以互補細菌的鉀離子運送能力。以Escherichia coli BL21(DE3) RIL異源表現膜蛋白KvMp,利用超高速離心以及界面活性劑dodecyl-β-D-maltoside可成功萃取約30 % 膜蛋白KvMp,將來可利用size exclusion chromatography純化KvMp蛋白並構築於脂質體(liposome),以分析KvMp蛋白in vitro活性。此研究是首次探討高鹽甲烷太古生物鉀離子通道。

Potassium is the major cation in all cells and plays a role in osmotic regulation and pH homeostasis. Potassium channel is a rapid potassium transport system to maintain the intracellular potassium concentration. Halophilic methanoarchaeon Methano- halophilus portucalensis FDF1T could grow at environment with salinity up to 2.9 M NaCl by accumulating 1.13 M of potassium ions and organic solutes as osmoprotectants. In this study, the complete gene of potassium channel (kvmp) was identified by metagenomic pyrosequencing. The secondary structure prediction revealed that KvMp consists of six transmembrane helices (TM) that is surrounded with the selectivity filter. In addition, the conserved positive charge amino acids (RxxRxxRxxRxxK) as voltage sensor in the fourth TM suggested that KvMp is a voltage activated potassium channel. In vivo activity of KvMp was demonstrated by the heterologous expression of kvmp gene in K+ uptake deficient mutant E. coli LB2003, which could not grow at low potassium condition (<10 mM). Campare with potassium transport deficient mutant E. coli LB2003, kvmp containing E. coli LB2003 could grow in low potassium (0, 1, 10 mM) environment. This result confirmed that KvMp is a potassium channel and archaeal potassium channel could complement the K+ transport activity in E. coli. In addition, the recombinant KvMp were heterologously expressed in E. coli BL21 (DE3) RIL. The membrane fraction of recombinant KvMp was obtained through ultracentrifugation and was further extracted by dodecyl-β-D-maltoside. The soluble KvMp were partially purified by Ni2+-NTA affinity chromatography. This is the first study of the voltage potassium channel in halophilic methanoarchaea.
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