人类重组PIF1蛋白的表达纯化和解螺旋酶活性的分析-微生物学报.PDF

生 物 工 程 学 报 Chin J Biotech 2009, February 25; 25(2): 268-273 Chinese Journal of Biotechnology ISSN 1000-3061 cjb@ © 2009 Institute of Microbiology, CAS CSM, All rights reserved 医学与免疫生物技术 人类重组PIF1 蛋白的表达纯化和解螺旋酶活性的分析 顾永清1,2 1 石河子大学医学院, 石河子 832002 2 Department of Experimental Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan 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。 关键词: PIF1 解螺旋酶, 蛋白表达和纯化, ATP 酶活性, 解螺旋酶活性 Overexpression, purification and helicase activity analysis of recombinant human PIF1 protein Yongqing Gu1,2 1School of Medicine, Shihezi University, Shihezi 832002, China 2 Department of Experimental Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 7348553, Japan 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