Expression,Purification And Enzyme Activity Analysis Of ClDHX36 Helicase

Helicases are a class of molecular motor proteins that use the energy provided by NTP to open hydrogen bonds between DNA or RNA.The helicase DHX36 is also called RHAU or G4R1,which belongs to a class of RNA helicases in the DEAH subfamily of the SF2superfamily.Since DHX36 was isolated from Hela cell lysates in 2004,the helicase has received widespread attention.Previous studies have found that DHX36 is involved in important life activities such as mammalian spermatogonia differentiation,hematopoiesis and heart development,neuronal precursor micro RNA dendritic positioning,and it is related to the synthesis and degradation of m RNA in multiple parts of cells.In addition,DHX36 also plays an important role in viral immunity.DHX36 in plasma cell dendritic cells mediates cytokine responses by specifically recognizing and binding guanine-rich sequences.DHX36 performs a variety of specific biological functions in the body,which means that it has a certain preference for the substrate structure and substrate sequence,but relevant enzyme activity data have not been reported.In this experiment,Columba livia DHX36helicase?Cl DHX36?was selected as the research object,fluorescence anisotropy and fast stop current fluorescence resonance energy transfer technique were used to systematically analyze the enzymatic activity from three aspects:substrate binding preference,unwinding conditions in vitro,and unwinding characteristics,provided a reference for the working mechanism of the DEAH subfamily helicase.Through sequence comparison,we found that Cl DHX36 has much lower sequence homology with the Bos DHX36 and h DHX36 at the N-terminus,we excised the N-terminus of the full-length protein with a specific proteolytic enzyme,the changes of enzymatic activity of the protein after digestion were measured.We finally found the following results:?1?The recombinant plasmid p ET15b-sumo-Cl DHX36 was successfully constructed,and induced expression in E.coli C2566H by IPTG to obtain soluble target protein.Cl DHX36 was purified by affinity chromatography and cation exchange chromatography.The aggregation state and homogeneity of Cl DHX36 were analyzed.It was found that Cl DHX36 existed as stable monomer in solution.?2?Cl DHX36 had a preference for the structure of the substrate,it had excellent binding activity for ss RNA,ss DNA and G4 DNA,but had poor binding activity for Fork and Bubble,the protein displayed the worst binding capacity with blunt-end ds DNA;Cl DHX36 also had a preference for the sequence composition of the substrate,it tended to bind G-rich DNA poly GT6;.?3?Excessive Cl DHX36 and tail-chain G4 substrate complex in vitro performed a dimer state.?4?The optimal in vitro unwinding conditions of Cl DHX36 were obtained.20 m M Tris-HCl p H 7.5,50 m M Na Cl,1 m M Mg Cl₂,2 m M DTT,reaction temperature 37?,protein concentration 20 n M.?5?Compared the unwinding activity of Cl DHX36 for different substrates and analyzed the unwinding characteristics.The results showed that the unwinding polarity of Cl DHX36was 3'-5',and Cl DHX36 was more inclined to unwind the double-stranded DNA substrate with Grich or G4 tail chain.Interestingly,the unwinding activities of Cl DHX36 were negatively correlated with the length of the substrate tails.?6?Cl DHX36^?Nwas obtained by cutting N-terminal of Cl DHX36 through specific proteolytic enzyme,compared with the full-length Cl DHX36,the binding activity of Cl DHX36^?N for G-rich DNA was not significantly different,but for G4 DNA was obviously decreased.Finally,the change of Cl DHX36^?N unwinding activity was analyzed,it was found that deletion of the N-terminal sequence made Cl DHX36 almost lose the ability to unwind G4DNA.In summary,Cl DHX36 protein was purified in this experiment,the substrate binding preference and unwinding characteristics of Cl DHX36 were analyzed,and the optimal unwinding conditions in vitro were optimized.It was clear that although the N-terminal sequences of Cl DHX36,h DHX36 and Bos DHX36 were quite different,their N-terminals were all related to binding and unwinding G4.