Protein Expression And Activity Analysis Of DHX9 Throtase In Drosophila Melanogaster

Racemase is widely distributed in various organisms and can use the energy released by ATP to open the hydrogen bond structure between the double strands of nucleic acid substrate.DHX9 hydrolase,also known as MLE,is a ATP-dependent RNA hydrocyclase,which can be used to open the hydrogen bond structure between the double strands of nucleic acid substrate.It can not only open double-stranded RNA but also unscrew double-stranded DNA,in retrovirus RNA activity,transcription,translation,splicing of mRNA precursors,MRNA output and ribosome formation play an important role.DHX9 racemase was first certified as a RNA lyase related to dose compensation of sex chromosome(X chromosome),and it was found that it was found to be in a variety of DHX9 can be used as a diagnostic marker and a potential therapeutic target for the treatment of NSCLC,which provides a new solution for solving some human diseases.In this study,the high purity DmDHX9 protein was obtained by prokaryotic expression system using RNA hydrocyclase DHX9(DmDHX9)of Drosophila melanogaster as experimental material.The effects of solution and temperature conditions on the binding ability of protein substrate were studied by fluorescence anisotropy.And substrate binding preference analysis.Then the different double chain ability of DHX9 was analyzed by stopping current spectroscopy.Finally,the composite verification,purification and preliminary screening of the crystallization conditions of DmDHX9 and DNA substrate were carried out.Through the analysis of the activity of DmDHX9 racemase,we can deepen our analysis of the activity of DHX9 hydrolysase family.It is helpful for us to study the interaction between DmDHX9 hydrocyclase and substrate and the analysis of crystal structure of the complex.The following are the main results of our research:1)DmDHX9 hydrolysase could be expressed in a large number of RosseTA(DE3)expressing strains,and a large number of target proteins were obtained in the supernatant solution after high speed centrifugation of broken bacteria.2)The target protein was obtained by Ni-NTA,which was eluted by HisTrap Q HP column gradient eluting,cut by 3C enzyme,and removed by reverse hanging with EDTA Ni-NTA resistance.The protein with high purity(more than 95%)waspurified by molecular sieves Superdex 200 column gel filtration.The molecular weight of protein is 118 kDa,isoelectric point 6.92.3)When the optimum conditions of DmDHX9 in vitro were determined,the concentration of NaCl,the concentration of DmDHX9,the condition of pH and the condition of temperature were investigated.The optimum conditions were determined as follows: 20 mM Tris-Hcl 7.0,25 mM NaCl,50 nM DmDHX9,2 mM MgCl2,reaction temperature 37 ?.4)When we explore the best substrate energy donor of DmDHX9 lyase,we find that the four energy donors(ATP/GTP/UTP/CTP)can be used by both DNA and RNA,but the highest utilization efficiency is CTP,.It reached(0.45 s ? 1 and3.0 s ? 1),respectively.This indicates that both DNA and RNA can use four kinds of energy donors(ATP/GTP/UTP/CTP)to complete the desorption reaction,and the best energy donors for the hydrolysis of the two substrate by DmDHX9 lyase are as follows: RNA or RNA can use four kinds of energy donors(ATP/GTP/UTP/CTP)to complete the demethylation reaction,and the best energy donors for the decomposing of the two substrates are as follows.CTP.5)From the three aspects of substrate desorption ratio,binding ratio and desorption rate,it was found that DmDHX9 racemase had obvious preference for DNA substrate with different tail chain structure,preferred to bind to 12bp-12 Grich,followed by 12bp-3G4.6)Using dsRNA(dsRNA-12p-10 polyU,dsRNA-12p-15 polyU,dsRNA-12p-20 polyU,dsRNA-12p-3G4-10polyU)as substrate,By comparing the difference of desorption ratio and desorption rate of different length tail chain structure substrate with the increase of substrate tail chain length,it was found that the desorption ability of DmDHX9 hydrolase became weaker with the increase of substrate tail chain length,and the G4 structure would decrease the desorption ratio of G4 structure,and the desorption ratio of G4 structure would be decreased with the increase of substrate tail chain length.However,the desorption rate did not decrease,but increased slightly..7)DNA substrate(3G4,12 ntGrich)and DmDHX9 complex were successfully obtained in buffer(20mM Tris-Hcl 7.0100 mM KCl,2mM MgCl2)and verified by(Native-PAGE)detection.8)The ssDNA substrate 12 ntGrich and DmDHX9 complex were screened for crystal,and the preliminary crystallization conditions were obtained.