Cloning, Expression and Functional Characterization of Potassium Channel from Haloterrigena sp. H13

• Main Authors: Ching-Yi Wu, 吳靜儀
• Other Authors: 賴美津
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/33978176817431509540

碩士 === 國立中興大學 === 生命科學系所 === 99 === The regulation of intracellular potassium concentrations by potassium channels is ubiquitous among all living organisms to maintain internal osmotic pressure and cellular homeostasis. The extreme halophilic archaeon Haloterrigena sp. H13 could grow at hypersaline environments with above 4 M or saturated NaCl condotions by accumulate nearly equal molar of potassium concentration inside the cell as compatible solute (osmolyte) to maintain the osmolarity cross the cytoplasmic membrane and stabilize proteins. In this study, the partial coding sequence (CDS) encoded the potassium channel (htgK) was identified in the genomic shotgun libraries of Haloterrigena sp. H13 was used as probe to screen and further obtained the full length htgK gene by Southern blot and cloning. Secondary structure prediction and functional motif search indicated that the N-terminus of the protein HtgK consists of two putative transmembrane helices that surround with the selectivity filter, one RCK domain (regulators of K+ conductance) and one KTN domain (K+ transport, nucleotide-binding) locate at the cytoplasmic C-terminus. The His-tagged HtgK, HtgKRCK, HtgKKTN and HtgKRCKKTN were constructed and heteroexpressed in E. coli BL21(DE3)RIL and purified using the Ni2+-NTA affinity chromatography. The truncated proteins of HtgK, HtgKKTN and HtgKRCKKTN were detected. To reduce the surface area of liquid culture level which may reduce the oxygen concentration could successfully reduced the amount of truncated proteins. The fluorescence spectroscopic studies showed that both Mg2+ and Ca2+ can stabilize the HtgKRCKKTN structure under denaturing condition. Through the ultracentrifugation and CHAPS extraction, only 10.9 % expressed HtgK was inserted into the membrane of E. coli BL21(DE3)RIL. The difference in membrane compositions between E. coli and extreme halophile and also the His tag in overexpressed HtgK proteins may affect the membrane insertion. To investigate the potassium transport activity of HtgK, HtgK was expressed heterologously in K+ uptake deficient E. coli LB2003 and the survival rate and growth rate in low potassium media were tested. On both 10 mM K+ added soild and liguid media, the survival rate and growth rate of the HtgK expressed cells was better than the host E. coli LB2003 and the cells with vector pTrc99A which indicated the complementation of potassium transport activity of archaeal HtgK to E. coli. This is the first study in the potassium channel of extreme halophilic archaeon.