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.
| Identifer | oai:union.ndltd.org:NSYSU/oai:NSYSU:etd-0202104-150605 |
| Date | 02 February 2004 |
| Creators | Shia, Hui-Ling |
| Contributors | Steven. S.-L. Li, none, none |
| Publisher | NSYSU |
| Source Sets | NSYSU Electronic Thesis and Dissertation Archive |
| Language | Cholon |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lib.nsysu.edu.tw/ETD-db/ETD-search/view_etd?URN=etd-0202104-150605 |
| Rights | restricted, Copyright information available at source archive |
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