Construction,Preparation And Characterization Of Linear SUMO2/3 Chains

SUMOylation is an important protein post-translational modification system in cells.SUMOs are small proteins with a molecular weight of about 11 k Da.The C-terminal carboxyl group of the SUMO molecule and the Lys residue on the substrate protein are catalyzed by the cascade reaction of three types of ubiquitination-related enzymes: E1 activating enzyme,E2 binding enzyme and E3 ligase,throught a covalent ligation to form isopeptide bonds,this process can be reversed by de SUMOase.The N-terminus of SUMO2/3 has a ΨKXE structure,which can be used as the attachment site of SUMO to form a poly-SUMO chain.SUMO modification plays an indispensable role in DNA damage repair、 cell cycle regulation、tumorigenesis、inflammation、neurodegenerative diseases and other physiological processes.There are 8 types of multimerized ubiquitin chains,of which one is the linear(M1)ubiquitin chain,and this type has not been found in the multimerized SUMO chain so far.Based on the correlation between ubiquitin and ubiquitin-like SUMO and the characteristics of linear ubiquitin chains,this study established a linear SUMO chain construction strategy to provide research materials for SENP enzyme activity analysis,SENP and SUMO substrate interaction studies.In this study,two basic plasmids p MD18TM-SUMO2/3-1st and p MD18TM-SUMO2/3-2nd.To extend the linear SUMO chain,p MD18TM-SUMO2/3-2nd was digested with Sac I(restriction site "GAGCT │C")and then T4 DNA polymerase was used blunt the sticky end of Sac I digestion,and Hind III cut this fragment to obtain SUMO2/3 gene fragment "C-SUMO2/3".This fragment was subcloned into the p MD18TM-SUMO2/3-1st vector I which digested with Stu I andand Hind III,then generated a linear SUMO chain with two SUMO2/3 genes,termed SUMO2/3-L2.Repeated these steps to generate longer linear SUMO chain with unlimited length theroetically.In this study,prokaryotic and eukaryotic expression vectors of linear SUMO2/3 chain genes with 2 to 4 SUMO lengths were constructed.Our results proved the feasibility of this strategy.These linear SUMO chains were expressed and purified using the prokaryotic expression system and Strep Tactin column.The activity of these linear SUMO chain proteins were characterized with de SUMOase SENP1/2.The results showed that linear SUMO2/3 chains with different lengths could be expressed in E.coli;SENP1/2 was able to hydrolyze these linear SUMO2/3 chains.These results indicate that the linear SUMO2/3 chain constructed and prepared in this study is active,which provides a new substrate for SENP and lays a foundation for further research on the linear SUMO2/3 chain in the future.