| Summary: | 碩士 === 國立交通大學 === 生物科技研究所 === 89 === Mammalian pancreatic group I phospholipase A2 (pPLA2-I) has its specific receptor on a variety of mammalian cells and various biological responses, e.g. synthesis of prostaglandins, fertilization, cell proliferation, smooth muscle contraction and inflammatory response, are elicited by PLA2-I via its receptor. For understanding molecular mechanisms leading to these cellular responses mediated by PLA2-I receptor, we try to develop a new, handy, safe and non-radioactive detection system. Avidin-biotin interaction technique provides a simple and sensitive detection method and is widely used. We constructed a expression vector pGEX-5X/PB encoding porcine pPLA2-I cDNA fragment with a truncated-biotinyl-carboxylase carrier protein (BCCP) fusion tag. The fusion protein was over-expressed in Escherichia coli BL21(DE3) and formed insoluble inclusion bodies. To improve fusion protein solubility, various expression conditions of the fusion protein were tested. In M9 minimum medium and 16℃ culture, little amount of soluble fusion protein was detected by western blotting analysis. After purification of bacterial lysates using the affinity column, 50 percent purity fusion protein was collected. The activity assay suggests that the fusion protein is functional. Though the biotinylation detection of insoluble fusion protein was successful, purified fusion protein couldn’t be seen.
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