Prokaryotic Expression Vector Construction, Expression And Characterization Of Human Ubiquitin Gene

Ubiquitin(Ub), a 76 amino acid residue protein, is the most highly conserved protein. Previous research shows that, through covalently modification by ubiquitin or ubls, the substrate proteins can be regulated in many different ways like stability, subcellular localization, enzymatic activity, protein-protein interaction, etc.The abnormal degration of protein is close to many disease,with the advancement of physic molecule biology technique,we can recognize and discuss the connection between them in molecule mechanism。Until now, more and more scientists are getting interested and participating in proteomics research of ubiquitin/Ubl modifications.Six pairs of oligonucleotide primers are designed by Primer5.0 software according to the Published Sequence of ubiquitin's gene,the target DNA fragment was abtained by overlap-1igation PCR. Expression vector pGEX-4T-Ub was constructed after the successful cloning of human ubiquitin, followed by prokaryotic expression and protein purification. Products got from this research will be used for further study of Ub. See results as following:1.according to the Published Sequence of ubiquitin's gene M17524 ,special primers were designed and 228bp fragment was abtained by overlap-1igation PCR.2. By the strategy of directional cloning,Ub was cloned directionally into the prokaryotic expression vector pGEX-4T-1. the recombinant plasmid was identified and analyzed by double digestion with retriction enzymes (BamH I) and SalI)and sequencing ,It is indicated that the Ub gene has been cloned successfully .3.The recombinant plasmid pGEX-4T-Ub was transformed into E.coli BL21(DE3) pLysS competent cells.The bacterium was induced by IPTG(1 mmol/L)and analyzed by SDS-PAGE,approximately 35kD exogenous protein was observed on the SDS-PAGE. 4. We get an induced expression product band,which correspond to the protein sizes of Ub,reported in the literature through western-blot.