| Summary: | 碩士 === 東海大學 === 化學工程與材料工程學系 === 107 === DNA mismatch repair plays an important role for preventing gene mutation. Any genetic mutation may lead to cancer. In prokaryotes and eukaryotes, guanine is very easy attacked by ROS to form 8-oxoG. The 8-oxoG lesion will lead to convert G:C to T:A and this is irreversible. In order to avoid the damage by 8-oxoG lesion, the organism has a corresponding preventive mechanism. In E.coli, base-excision repair is the major mechanism to remove oxidized guanine by specific DNA glycosylases MutT、MutM and MutY.
In this study, we ligase MutY gene to pET21a(+) vector and pSEI vector by genetic recombination, respectively. The plasmids transformed to BL21(DE3), and expressed MutY by IPTG. In the system pET21a(+), the overexpression of protein will lead to the formation of inclusion body. By analyzing the relationship between inclusion bodies and the variation of inclusion body during denaturation, we conjecture the inclusion body may be gradually coated by the target protein and the outer layer is the other protein. Because pSEI plasmid containing the sumo, the MutY protein is soluble. The fusion protein containing EGFP, this characteristic may lead to better purification. The pure fusion protein can facilitatation for large applications.
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