| Summary: | 碩士 === 國立中興大學 === 生物科技學研究所 === 94 === Abstract
Bamboo mosaic virus (BaMV) belongs to the alphavirus-like superfamily. The genome of BaMV is a positive-sense RNA. The ORF1 encodes a 155-kDa replicase that contains capping enzyme, RNA 5΄-triphosphatase and the RNA-dependent RNA polymerase domain (RdRp) from N. to C-termini. Previously, the capping enzyme was expressed in E.coli cell but it was inactive and was found as inclusion body and was still inactive after lots of refolding test. Hence, we expressed this protein in yeast (Saccharomyces cerevisiae) now. Biochemical studies demonstrated that the capping enzyme possess both enzymatic activities of m7GMP:mRNA AdoMet-dependent guanylyltransferase and GTP methyltransferase. Nevertheless, the capping enzyme produced by yeast was associated with membrane and the expression level of capping enzyme was extremely low which made it difficult to purify. Previous studies in our lab show that the anionic detergent, sarkosyl, is the most efficient extraction agent for this membrane protein. However, the purity and yield of capping enzyme purified with sarkosyl is not enough for further studies on catalytic mechanism or structure of this enzyme. The main aim of this study is to create a more efficient purification method for further analysis. By altering various conditions it was found that this enzyme can be extracted by 1 M KCl with 2% Triton X-100 and 20% glycerol. The capping enzyme with higher specific activity can be harvested from the membrane fraction by passing the extraction fraction through a metal affinity chromatography (Ni-NTA) column and eluting it with the elution buffer containing 0.8% OTG and 20% glycerol and 250mM imidazole. The quality of the capping enzyme purified by this new method is improving. Compared with the capping enzyme purified by sarkosyl containing purification, the specific activity, purity and recovery of the capping enzyme purified by this new method can increase 1.7, 1.6, 1.8 fold respectively. Therefore catalytic mechanism or structure analysis of capping enzyme can be anticipated by this efficient purification method in the future.
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