Molecular studies on the interaction between human post-translational modifier protein SUMO and centromere protein CENP-C

• Main Authors: Hui-Ling Shia, 夏慧伶
• Other Authors: Steven. S.-L. Li
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/16697629663598776437

碩士 === 國立中山大學 === 生物醫學科學研究所 === 92 === Human post-translational protein modifier protein SUMO-1/2/3 genes code for proteins homologous to yeast SMT3 protein, which is encoded by a suppressor 3 of MIF2 mutation in centromere protein gene. The yeast MIF2 protein shares at least two regions of similarity with mammalian centromere protein CENP-C. It would be of interest to investigate the possible interaction(s) between human CENP-C and SUMO-1/2/3 proteins. A CENP-C cDNA fragment was cloned using RT-PCR with total RNAs form Hela cells. This cDNA fragment encoding CENP-C amino acids 342-764 (MW 38 kDa designated C38) was tagged with EGFP. The sub-cellular localization and in vivo sumoylation in HeLa cells were carried out. The EGFP-C38 protein was shown to co-localize with active forms of Flag-SUMO-1/2/3GG proteins in nucleus of Hela cells. The EGFP -C38 protein was also shown co-immunoprecipitated with antibodies against SUMO proteins. The protein conjugates were analyzed on SDS-PAGE and their western blots were probed with either anti-GFP or anti- Flag antibodies. The molecular weight of EGFP-C38 protein was found to be higher than the expected MW, indicating that EGFP-C38 protein was sumoylized. This part ( 333 amino acids) of CENP-C protein (943 amino acids) was expressed and purified. The in vitro sumoylized His-C38 protein fragment was analyzed on SDS-PAGE, and the western blot was probed with either anti-SUMO-1 or anti-SUMO-2 antibodies. The C38-His protein fragment appeared to be sumoylized, and the isopeptide bond between the C-terminal glycine of SUMO and lysine of His-C38 was analyzed by MALDI-TOF-TOF. C38 cDNA was sub-fragmented into C28 and C10 fragments transformed to BL21 strain for expression protein and purified protein. S-tagged SUMO-1/2GG modify C28-His and C10-His fragments, the isopeptide bond between the C-terminal glycine of S-tagged SUMO-2GG and lysine of C10-His was identified analyzed by MALDI-TOF-TOF. The isopeptide bonds between either S-tagged SUMO-1GG and C28-His or S-tagged SUMO-1/2GG and C28-His /C10-His are being analyzed.