Expression and purification of the Kir6.2 channels

碩士 === 國立臺灣大學 === 口腔生物科學研究所 === 93 === 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 functio...

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Bibliographic Details
Main Authors: Pi-Jung Hsu, 許碧絨
Other Authors: Kuo-Long Lou
Format: Others
Language:zh-TW
Published: 2005
Online Access:http://ndltd.ncl.edu.tw/handle/92281665146905793645
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Summary:碩士 === 國立臺灣大學 === 口腔生物科學研究所 === 93 === 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.