Expression And Purification Of Recombinant Human DNA-PKcs Truncated Protein In E.coli

DNA-PKcs as a catalytic subunit and two regulatory subunits Ku70/Ku80 heterodimer to form DNA-dependent protein kinase(DNA-PK).This kinase belongs to the phosphatidylinositol-3 kinase-related kinase family(phosphatidylinositol3-kinase-relatedkinases,PIKK)and has serine / threonine(Ser/Thr)kinase activity.It is a key protein kinase in the process of genomic DNA damage repair,which participates in and determines the whole process of non-homologous terminal connection of(NHEJ)DNA damage repair pathway.Among them,DNA-PKcs,the core member of DNA-PK,plays an important role in the repair of DNA double-strand break(DSBs),maintenance of telomere and chromosome structure,apoptosis,tumor inhibition and so on.DNA-PKcs consists of 4128 amino acids.According to the research and analysis of structural biology,the full length of DNA-PKcs can be divided into three parts: the N-terminal(N-terminal),the circular scaffold region(CircularCradleunit,893-2801aa)and the head region(Headunit,2802-4128aa).The main functional domains of DNA-PKcs are distributed in the Headunit region,including FAT(2908 Mel 3539)domain,PI3K(3645-4029aa)kinase active domain and FAT-C domain.A large number of studies have reported that DNA-PKcs plays a leading role in the phosphorylation of serine at position 139 of histone H2 A.X in the process of DSBs response.Interestingly,the latest research reports suggest that the phosphokinase activity of DNA-PKcs may be involved in the process of replacing the histone variant H2 A.Z into nucleosomes.However,the areas involved in this replacement function are not clear.Therefore,in-depth study and clarification of this replacement mechanism of DNA-PKcs has important theoretical significance to clarify its biological function in cells.In view of the fact that DNA-PKcs is a macromolecule containing 4128 amino acids,in order to facilitate in vitro experiments and to clarify the functional region of the replacement histone variant H2 A.Z,in this study,the full-length DNA-PKcs was designed into eight regions: PI3K(3747-4128aa),FAT(2908-3539aa),1-412 aa,500-1025 aa,1000-1525 aa,1500-2000 aa,1878-2182 aa and 2261-2700 aa.Then,the eight truncated plasmids were introduced into the pGEX-6p-1 vector which can be expressed in E.coli,and the recombinant plasmid pGEX-6p-1-DNA-PKcs-truncate with GST fusion protein was constructed.Secondly,with the help of E.coli expression system in vitro,the expression conditions of different truncated body proteins were optimized,and different truncated body proteins of DNA-PKcs were purified by GST-pulldown,and the expression of truncated body proteins was confirmed by Coomassie brilliant blue and Western blotting.Finally,different truncated DNA-PKcs proteins were concentrated and purified by a separation column(pore size < 3000MV).After protein quantification,the in vitro phosphorylase activity detection system was used to determine the catalytic activity of the recombinant proteins by using histone H2 AX as substrate and specific antibody phosphorylated at position 139.The purpose of this study is to identify the functional regions with phosphokinase activity by expressing different truncated body proteins of DNA-PKcs,and to provide theoretical and experimental basis for further elucidating the interaction mechanism between DNA-PKcs phosphokinase activity and its replacement histone variant H2 A.Z.