The purification and preliminary functional study of N-terminal truncated human PIF1 helicases
- VernacularTitle:不含氨基末端的PIF1解螺旋酶的纯化和初步功能研究
- Author:
Yongqing GU
- Publication Type:Journal Article
- Keywords:
PIF1 helicase;
DNA purification;
ATPase
- From:
Journal of Xi'an Jiaotong University(Medical Sciences)
1981;0(03):-
- CountryChina
- Language:Chinese
-
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 fragment of human PIF1 from 540～1 923 was cloned from Hela cDNA library,and the recombinant PIF1△N protein was successfully overexpressed in E.coli.The purification procedure of PIF1△N protein was established and its biochemical activity was identified.Conclusion N-terminal truncated PIF1 helicase,PIF1△N,has ATPase activity,which is DNA and Mg2+ dependent.
