Enrichment Of Ubiquitinated Proteins And Their Interacting Proteins Using Hybrid Tandem UBD Domains

Protein post-translational modification is one of the most important mechanisms for regulating protein stability and function.At present,people have adopted various strategies to analyze the ubiquitinated proteome.Both ubiquitinated and ubiquitinated conjugates exist at lower levels.Although the ubiquitination modification belongs to a high-abundance type of post-translational modification,its content in the overall proteome is relatively low,accounting for only 1% or less of the total proteome.The action of 26S proteasome and deubiquitinating enzyme results in the ubiquitin chain being easily removed by catalysis.It is difficult to identify ubiquitin conjugates without pre-enriching low-abundance ubiquitinated proteins.Therefore,how to efficiently enrich and purify ubiquitinated proteins has become a key issue in the study of ubiquitination.In this test,a bait protein that can be combined with a polyubiquitinated chain is synthesized by the whole gene.The bait protein is composed of two repeated UBD(ubiquilin 2-derived UBA,UQ2,575-624 amino acids;RABGEF1-derived A20-ZnF,A20,9-73 amino acids)domains repeated twice,and the sequence is connected to the C-terminal of the GST tag It can express the bait protein fused with GST tag.The constructed recombinant plasmid was transferred into E.coli and IPTG induced protein expression.The induction conditions of recombinant protein GST-HzUBD were optimized from the induction concentration and induction time.The efficiency of two agarose resin coupling bait proteins and the enrichment effect of ubiquitinated protein were compared.The enriched samples are tested on the machine.The main results are as follows:(1)Induction with 0.1 mM IPTG at 16 ? for 12 h is the best induction condition for recombinant protein GST-HzUBD.The successfully induced recombinant protein GST-HzUBD was purified,and the purified and replaced bait protein was detected by Western Blot technology using GST tag antibody.The results showed that the bait protein fused with GST tag was successfully obtained.(2)The NHS activated agarose resin forms a stable covalent bond with the bait protein to prevent the elution of the bait protein in large quantities and can enrich more ubiquitinated proteins.MG132 inhibits the activity of 26S proteasome and deubiquitinating enzyme to increase the content of ubiquitinated protein.Through cell experiments,50 ?M MG132 was added to the experimental group,and the control group was added with the same amount of DMSO.Western Blot results showed that the content of ubiquitinated protein in the cells increased significantly after treatment with MG132.Compared with the control group,the experimental group was enriched more Ubiquitinated protein.(3)1786 proteins were detected by liquid chromatography-tandem mass spectrometry,and path analysis showed that ubiquitination modification not only mediates protein degradation,but also participates in cell apoptosis,DNA damage repair,and transcription regulation.,Immune response,cell cycle and many other cellular process functions.Since this experiment is an enrichment process using hybrid tandem domains under non-denaturing conditions,in addition to enriching ubiquitinated proteins,it also enriches proteins that interact with ubiquitinated proteins,such as E3 Ligase etc.This experiment provides a method foundation for the systematic study of the ubiquitination modification involved in various biological processes.