Free amino terminal of the PIF1 helicase function of purification and preliminary.docVIP

 PAGE \* MERGEFORMAT 12 Free amino terminal of the PIF1 helicase function of purification and preliminary [Abstract] described the molecular level the purpose of human PIF1 helicase physiological function, purification contained only helicase motifs without N at the end of the PIF1 protein - PIF1 N, and its biochemical activity were detected. Methods Hela cell cDNA library as template, PCR amplified PIF1 cDNA 540 ~ 1923 of the sequence of the cDNA 5 ‘end of the introduction of six-histidine tag inserted into pET20b expression vector, the recombinant plasmid pET20 PIF1 N. by a total of into the recombinant plasmid and a plasmid encoding rare rRNA, PIF1 N protein was expressed in E. coli. at 4 through a series of affinity chromatography, purified by high performance liquid purification system PIF1 N protein, and to detect its biochemical activity. Results from the Hela cell cDNA library cloned PIF1 protein gene fragment of 540 ~ 1923, it successfully expressed in E. coli. establish PIF1 N protein purified by affinity chromatography and detection their ATP activity. Conclusion PIF1 protein without N terminal-PIF1 N, has a dependency on magnesium ion and DNA in the ATP activity. [Keywords:] PIF1 helicase ATP enzyme protein purification ABSTRACT: Objective To elucidate physiological functions of human PIF1 helicase at the molecular level, purify N terminal truncated PIF1 helicase, PIF1 N, and assay its biochemical properties. Methods The N terminal cDNA sequence of PIF1 helicase was amplified by PCR using the Hela cell cDNA library as template. The cDNA with a histidine tag at the N terminus was inserted into the pET20b vector to produce recombinant plasmid. The recombinant PIF1 N was successfully expressed by co transforming a plasmid encoding rare rRNA. At 4 through a series of affinity column the recombinant PIF1 N protein was purified by fast protein liquid chromatograph. The biochemical activity of PIF1 N was assayed. Results The cDNA fragm