Expression, Purification And Activity Analysis Of A Pif1 Helicase From Anaerobaculum Hydrogeniformans

Helicase is catalyzed by energy donor move in a certain direction and open the hydrogen bonds of the base substrate nucleic acid. And it is required in many important physiological activities of organisms. Pif1 family helicases function as 5’ to 3’ DNA helicases catalyzed by adenosine triphosphate(ATP). It is widely concerned since the first discovery. They play important roles in the replication of telomeric DNA, mitochondria DNA, and the processing of DNA Okazaki fragments. More important is the pif1 helicase impact telomerase have been verified in vitro and in vivo and this may develop a new ways to inhibit the tumor telomerase activity in order to cure tumor. So it is of great significance for the study of Pif1 helicase of biological evolution research and control of human diseases. We aim to obtain the Pif1 helicase from Anaerobaculum hydrogeniformans(Ana Pif1) by prokaryotic expression system,and explore the optimal conditions for Ana Pif1 to display unwinding activity in vitro and aggregation state by stopped-flow and fluorescence resonance energy transfer(FRET) and Dynamic light scattering(DLS) technique. The following results were obtained.1) We synthesized the entire gene of Ana Pif1 and constructed a recombinant plasmid p ET15b-sumo-Ana Pif1. After the identification of the correct then we transformed the plasmid into Escherichia coli host strain BL21(DE3). The full-length Ana Pif1 helicase was expressed of large number with good solubility.2) Target protein in the supernatant was purified through Ni-NTA affinity chromatography, cleaved by Sumo protease and followed by Heparin chromatography with FPLC system. Finally we obtained the full-length Ana Pif1 helicase with high purity(>95%).w M 60 k Da, p I 6.89.3) In order to obtained the optimal buffer conditions, we change the p H condition, the concentration of Tris、Na Cl、Mg Cl2 and DTT. Finally we determined the optimalbuffer conditions were Tris-HCl(p H 7.0) 20 mmol/L, Na Cl 20 mmol/L, Mg Cl2 2mmol/L.4) Ana Pif1 is a kind of thermophilic protein so it’s optimal unwinding condition must depend on specific temperature. We setted a series of temperature gradient(25-50 ℃)in optimal buffer condition. Results showed that the unwinding activity strongly depended on temperature. High or low temperature both was not conducive to the unwinding reaction and 37 ℃was the optimal condition.5) This study found that Ana Pif1 could use four kinds of nucleoside triphosphate as its energy donors. But the utilization rate had obvious difference. The unwinding rate(1.4 s-1) and amplitude(50%) were significantly higher in the presence of ATP. This noted that ATP was the most suitable energy donor.6) The study of Ana Pif1 unwond this three kinds of substrate 26 nt-17 bp、50 nt-16 bp and 26 nt-3G4-17 bp we could draw the conclusion that Ana Pif1 had no obvious preference for DNA substrates such as G4-DNA and double-stranded DNA. Because the unwinding rate(the three were all between 0.95-1.10 s-1) and amplitude(the three were all between 40-50%). This implies that Ana Pif1 could not be activated by G4-DNA just like Sc Pif1, and this propertiy was similar to Bs Pif1.7) By using Dynamic light scattering(DLS) technique in optimal unwinding condition,we foud the radius of Ana Pif1 was 3.271 and the w M was 60 k Da. This means that in this condition Ana Pif1 was a monomer. And this conclusion was also similar to Bs Pif1 but different with Sc Pif1.