人PIF1解螺旋酶克隆表达及2种细胞裂解法纯化PIF1蛋白比较

Cloning and expression of human PIF1 helicase and the comparison of two methods to lysis the cells

目的克隆、表达人类全长PIF1解螺旋酶,建立PIF1纯化方法。方法从Hela细胞的cDNA文库中PCR扩增得到PIF1解螺旋酶cDNA,插入N-末端融合有六聚组氨酸的表达载体pET15b产生pET15b-PIF1重组质粒,转化RosettaTM2(DE3)感受态细胞使PIF1蛋白在大肠杆菌中表达,分别用溶菌酶热裂解法和玻璃珠震荡法裂解大肠杆菌细胞,再用高效液相的镍亲和柱亲和层析纯化PIF1蛋白,比较2种方法释放的PIF1蛋白。结果克隆了PIF1基因,并使PIF1蛋白在大肠杆菌中得到成功表达,2种裂解法均能使PIF1蛋白释放出来,玻璃珠裂解法释放的PIF1蛋白较热裂解法高2倍。结论玻璃珠震荡法裂解细胞能释放更多的PIF1蛋白,是纯化PIF1蛋白的一个较好的选择。

Objective To clone and express human full-length PIF1 helicase and establish the method to purify human recombinant PIF1. Methods The full-length PIF1 cDNA was amplified by PCR from Hela cell eDNA library and inserted to pET15b with 6 X histidine tag at its N-terminus to form pET15b-PIF1 plasmid. The recombinant pET15b PIF1 plasmid was transformed to RosettaTM 2（DE3） and PIF1 protein was expressed. Heat lysis with lysozyme and vortex with glass beads were used to lysis cell to supply nickel affinity chromatograph by fast protein liquid chromatograph. The PIFI proteins released by two different methods were compared. Results The PIF1 gene was cloned and PIF1 protein was successfully expressed. Both of the above two methods can release PIF1 protein to the supernant, and PIF1 protein released in vortex with glass beads increased two times higher than that in heat lysis. Conclusion Vortex with glass beads can release more PIF1 protein to supernant and is a good option to purify PIF1 protein.