Expression, Purification And Functional Analysis Of The Thermodesulfovibrio Yellowstonii H. TyPif1

Pif1 helicase is a class of DNA helicase which can move along the DNA strand from 5’ to 3’ to unwind DNA strands. Pif1 helicase are found in all organisms, among which Saccharomyces cerevisiae, Schizosaccharomyces pombe, mouse and human were studied indepth. In vivo The activity of Pif1 indicates that it plays an essential role in maintaining the stability of chromosomal DNA, mitochondrial DNA and ribosomal DNA. The mutation of Pif1 gene leads to abnormal DNA recombination, disturbing the normal physiological activities of cells. Pif1 helicase help cells to recognize DSB(double strand breakage) region, repairing the breakage of double strand DNA. Furthermore, Pif1 helicase also acts on the telomeric region, suppressing the activity of telomerase, keeping cells staying in a normal life stage. Researches on activities of Pif1 were mostly truncated proteins, such as: truncated Saccharomyces cerevisiae Pif1(ScPif1) and truncated human Pif1(hPif1) in vivo. These studies found that Pif1 helicase has strict 5’-3 ’direction of the helicase polarity, and that the the ability of Pif1 helicase binding and unwinding G4 DNA is much higher than that of other helicase family members.Although researches on Pif1 helicase have achieved some achievements, the unwinding mechanism of Pif1 helicase is not yet clearly, in addition, full-length Pif1 helicase is difficult to get, thus, the understanding on Pif1 protein is limited. In this research we use prokaryotic expression system is used to obtain Pif1 helicase from Thermodesulfovibrio yellowstonii H.(TyPif1), and study its activity of DNA binding, unwinding and enzyme thermophilic characteristic in vivo.our research can provide useful help to study the mechanism of Pif1 helicase. Our results of this study are as follows:1. We synthesised codon-optimized Thermodesulfovibrio yellowstonii H. Pif1 gene, constructed a recombinant plasmid pET15b-SUMO-TyPif1, containing a SUMO tag gene which can help protein folding, and then transformed the plasmid into Escherichia coli host strain BL21(DE3).Soluble protein was preliminarily purification by Ni-NTA affinity chromatography, then SUMO protease was used to digest the recombinant protein, and then, using the Ni-NTA affinity chromatography to purify secondly, Heparin chromatography was used in the final step, all steps were operated at 4 ℃.Using the purification steps above all, About 10 mg full-length TyPif1 helicase with high purity(>95%) can be obtained in 1 L medium.2. By measuring the binding activity of different length of ss DNA with TyPif1 helicase, we found the binding size of TyPif1 was 11.27 nt. The results of TyPif1 binded different substrates indicated that the binding ability of TyPif1 with ssDNA and G-quadruplex(G4) DNA was much higher than dsDNA.3. We measured the CD absorption spectrum of TyPif1 helicase, the results showed that the secondary structure of TyPif1 helicase was relatively stable under 60 ℃.The results of unwinding activity indicated that the unwinding rate of TyPif1 increased from 0.09 s-1 to 0. 23 s-1 with the temperature raised, this result was consistent with the growth and metabolism of T.yellowstonii in its living environment.4. By comparing TyPif1 helicase unwinding ds DNA and G4 DNA, we found that the rate of TyPif1 unwinding G4 DNA is faster than ds DNA, and G4 DNA can stimulated the ability of unwinding ds DNA.Our research established the expression and purification system of TyPif1 helicase. By measuring its activity of binding and unwinding, we have a knowledge of the optimal binding substrate and unwinding substrate.The conclusion about our research can provide a reference for further studying the unwinding mechanism of TyPif1 helicase.