SEE1p of Saccharomyces cerevisiae:protein expression in Escherichia coli, purification and methylation activity

• Main Authors: Yan-Sheng Jian, 簡晏生
• Other Authors: Ming-Kai Chern
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/77586890085983434076

碩士 === 淡江大學 === 生命科學研究所碩士班 === 98 === As humen genome project is finished, the research of life science has changed its paradigm from genomics to proteomics gradually. The study of protein modifications and transcriptional regulation has started to dominate the research headlines. Protein methylation plays a central role in both of these fields, and it is a post-translational modification of frequent occurrence. Although in many cases the roles of protein methylation are poorly understood, some have been known to play regulatory roles in the cell. Up to now, there are still many protein methyltransferases for protein methylation that remains to be identified . The uncharacterized Saccharomyces cerevisiae open reading frame (ORF) YIL064w is predicted to encode a cytoplasmic 28 kDa protein,recognized by sequence similarity as a putative S-adenosyl-L-methionine methyltransferase. We constructed SEE1 into E. coli to express See1p . We used His-tag column to purify See1p. The protein extract from ΔSEE1 yeast strains was mixed with See1p and the cosubstrate S-adenosyl-L-methionine of which the methyl being transferred is radioactive. We used isoelectric focusing electrophoresis (IEF) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to isolate substrates being methylated. We then used MALDI-TOF to identify the substrates.