Expression,Purification And Activity Study Of Human And Porcine RAD51 Recombinant Protein

DNA repair protein RAD51（DNA repair protein RAD51 homolog 1）is a key protein in eukaryotes that performs the homologous recombination repair pathway,exhibiting DNA-dependent ATPase activity upon binding to single-stranded DNA（ss DNA）and double-stranded DNA（ds DNA）.During homologous recombination repair,RAD51 proteins bind ss DNA to form nucleoprotein filaments.The protein will invade homologous DNA sequences to accurate repairing DNA homologous.This process plays a role in maintaining genomic stability and cancer therapy.Although numbers studies about RAD51 had been done,understanding functions and stabilities of RAD51 and its variations are important for biological resistances related DNA repairing.Herein,human RAD51（Homo sapiens RAD51,h RAD51）and porcine RAD51（Sus scrofa RAD51,s RAD51）were constructed into prokaryotic expression vectors.After optimized the expression and purification methods of h RAD51 and s RAD51 in E.coli expression system,the h RAD51 and s RAD51 with purity higher than 95%were obtained.Subsequently,the secondary structure,DNA binding activity,and ATPase activity of h RAD51 and s RAD51 were studied using circular dichroism spectroscopy,fluorescence anisotropy,and Na⁺K⁺-ATPase activity assay.The main results obtained from the experiments are as follows.（1）The synthetic sequence-optimized hrad51 and srad51 can be expressed in the prokaryotic expression system（E.coli）in large quantities,and the purification methods combined with Ni-NTA affinity chromatography column and Superdex 200size exclusion chromatography column,high purity h RAD51 and s RAD51 can be obtained.（2）The secondary structures of both h RAD51 and s RAD51 were greatly changed in p H4 citrate phosphate buffer.（3）h RAD51 showed strong Mg²⁺or Ca²⁺dependence and ATP dependence,and the binding activity increased with the increase of DNA length;s RAD51 did not show Mg²⁺,Ca²⁺or ATP dependence,and the binding activity also increased with the increase of DNA length.（4）The ATPase activity of h RAD51 was more affected when the environment was acidic at p H3.5 or alkaline at p H10,while the ATPase activity of s RAD51 was less affected;the ability of h RAD51 and s RAD51 to hydrolyze ATP was impaired when the final concentration of 3.4 m M Co²⁺or Mn²⁺was present in the environment.This study demonstrated and optimized the prokaryotic expression and purification methods of h RAD51 and s RAD51.The DNA substrate binding activity and ATPase activity of h RAD51 and s RAD51 under different environments were measured and analyzed,which laid the foundation for further research on the biological functions of h RAD51 and s RAD51 and the research work of endogenous s RAD51.