Optimized Expression and Purification of Recombinant Kir6.2 delta C26 Channel Protein

• Main Authors: Chun-Man Huang, 黃春滿
• Other Authors: Kuo-Long Lou
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/35500967384737861734

碩士 === 臺灣大學 === 口腔生物科學研究所 === 96 === ATP sensitive potassium (KATP) channels are hetero-octamer of two proteins: The K+-selective channel pore is forms with Kir6.x (inwardly rectifying potassium channels) and the regulatory subunit is composed of SUR (sulphonylurea receptors). They are distributed in various tissues include the pancreatic β-cell, cardiac and skeletal muscle are affected by intracellular ATP, PIP2 and MgATP. The topological and physiological characteristics of Kir6.2 are two transmembrane domains and transfer the K+ across the membrane and stabilizing the resting membrane potential. It related to the neuroprotection and loss-of-function in Kir6.2 cause permanent neonatal diabetes. Crystallization of Kir6.2 has not proved successful. The detail mechanism of the nucleotides regulating the channel activity and gating have not been defined so far. We attempted to express and purify the fusion proteins of Kir6.2 to determine the single crystal structure. Kir6.2 delta C26 was in-frame subcloned into pET20b, pET28a with hexhistidine tag in its N and/or C terminus to reduce the effect of the co-expressed protein and pGEX4T-1 vector with GST-tag to increase the solubility of fusion protein in various E. coli strains. Until now, it was found from the results that the target protein has not induced successful and maybe toxic to the E. coli host cell. Although glycosylation is not prerequisite for functional Kir6.2. In order to consider ATP sensitive potassium (KATP) channels which need highly regulation, we also try performance advanced expression system as: yeast and insect cell. Expression with yeast and inset cell is expected to decrease toxicity and increase the production. The data come out less protein was purified. Furthermore, we will try the gene replica insertion events and raise the transfection efficiency of the Baculovirus-insect cells system to obtain the pure protein in large-scale.