Study of the activity and substrates of recombinant ABP140p and NNT1p of Saccharomyces cerevisiae

• Main Authors: Chung-Yen Lee, 李政彥
• Other Authors: Ming-Kai Chern
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/89989258164839040209

碩士 === 淡江大學 === 生命科學研究所碩士班 === 97 === As humen genome project is finished, the research of life science has changed its paradigm from genomics to proteomic from genomic gradually. The study of protein modifications and transcriptional regulation has strated to dominate the research head -lines. Protein methylation plays a central role in both of these fields, and it is a post -translation 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 methyltransferase of protein methylation that remains to be identified . The sequences of ABP140 and NNT1 of Saccharomyces cerevisiae match the se -quences of methyltransferase in the database. In this study, we planned to find the activity and substrate of ABP140p and NNT1p. We constructed ABP140 and NNT1 into E. coli to express ABP140p and NNT1p. We used His-tag column to purify ABP140p and NNT1p. The activity is tested by a reaction containing ABP140p and NNT1p, protein extract from ΔABP140 and ΔNNT1 yeast strains 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 identified the substrates. The result showed the NNT1 is a protein methyltransferase. However our method can not see the activity of ABP140p.