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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 PIF1 purification method. Methods PIF1 helicase cDNA was amplified by PCR from Hela cell cDNA library. The recombinant plasmid pET15b-PIF1 was inserted into the expression vector pET15b with N-terminal hexa-histidine fusion and transformed into RosettaTM2 (DE3) competent cells to express PIF1 The protein was expressed in E.coli and lysed by lysozyme and glass beads respectively. The PIF1 protein was purified by high performance liquid phase affinity chromatography with nickel affinity column. The PIF1 protein released by the two methods was compared . Results The PIF1 gene was cloned and the PIF1 protein was successfully expressed in E. coli. Both of the two lysing methods released the PIF1 protein, and the PIF1 protein released by the glass bead lysis method was 2 times higher than that of the pyrolysis method. Conclusion Glass beads shock method can release more PIF1 protein, which is a better choice for purifying PIF1 protein.