Effect of SIM on the expression and purification of SUMOylated MTF-1

• Main Authors: Lin, Chang-Yi, 林昌儀
• Other Authors: Lin, Lih-Yuan
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/45994536984914255879

碩士 === 國立清華大學 === 分子與細胞生物研究所 === 98 === Metal-responsive transcriptional factor 1 (MTF-1) regulates a variety of genes involved in metal homeostasis and oxidative stress. Recently, we have demonstrated that MTF-1 can be SUMOylated and resulted in an alternation of the transcriptional activity. We attempted to express and purify SUMOylated MTF-1 for functional study. In our experiment, MTF-1 and the in vivo SUMOylation system were co-expressed into E. coli. Various conditions were examined to obtain the optimal production of the protein. However, purification of the SUMOylated product was unsuccessful since native MTF-1 consistently presented in the eluted fractions with the modified protein. Analysis of the MTF-1 primary sequence reveals a consensus SUMO-interaction motif (SIM) located at the carboxyl terminal region, which may interact with SUMO by hydrophobic interaction. Mutation at the SIM region caused the loss of interaction and thus SUMOylated MTF-1 can be isolated. This result susggests a cross-interaction of MTF-1 and its SUMO-conjugated product in the cells. DNA-binding activity of the native MTF-1 and SIM mutant was analyzed by electrophoretic mobility shift assay. Native MTF-1 binds DNA only when cell extract is present. However, SUMOylated MTF-1 can form complex directly with DNA. The result implies a formation of special conformation after SUMO modification that allows the MTF-1 to react with DNA.