人类重组PIF1蛋白的表达纯化和解螺旋酶活性的分析

Overexpression, purification and helicase activity analysis of recombinant human PIF1 protein

摘要:

Pif1解螺旋酶家族在酵母到人的进化中非常保守, 在生物体内具有很多重要的生理作用。为了从生物化学水平研究人类PIF1的功能, 从HeLa细胞的cDNA文库中克隆得到人类PIF1全长基因, 通过共转化一个携带稀有遗传密码tRNA1的质粒和一个编码分子伴侣的质粒, 增加了PIF1蛋白在大肠杆菌中的表达, 最后通过快速液相色谱纯化系统, 采用亲和层析和凝胶过滤, 纯化了人类重组PIF1蛋白。生物化学活性检测证明了纯化的人类PIF1蛋白具有ATP酶及解螺旋酶活性。人类PIF1蛋白的纯化为我们从分子水平理解PIF1在体内的功能奠定了基础。

Abstract:

Pif1 subfamily helicase is conserved from yeast to humans with a lot of cellular functions. In order to elucidate the function of human PIF1 helicase from biochemical level, we cloned human PIF1 gene by PCR from HeLa cell cDNA library. We co-transformed a pMStRNA1 plasmid encoding rare tRNA codons and a plasmid encoding molecular chaperon to greatly enhance the overexpression of human PIF1 protein. Finally we purified full-length PIF1 helicase by column chromatograph carried out at 4°C using fast protein liquid chromatograph (FPLC) system. The human PIF1 protein was purified in enough quantity for detailed biochemical analysis. Biochemical assay showed that PIF1 had ATPase activity and helicase activity. The purification and biochemical properties analysis of human PIF1 helicase will allow us to understand how, at the molecular and mechanistic level, this conserved helicase operate in the cell.