E2,E3and DUB Gene Deletion In Saccharomyces Cerevisiae And The Expression And Purification Of Ubiquitin-binding Domains

In eukaryotic cells, the selective degradation of many proteins is carried out byubiquitin-proteasome system (UPS). In this pathway, the ubiquitin is activated by theubiquitin-activating enzyme (E1) and then be transferred to the ubiquitin-conjugatingenzyme (E2). The ubiquitin ligase (E3) which recognizes the targeted proteins and E2,transfers the ubiquitin to the targeted proteins. The targeted proteins covalently attached byubiquitin are recognized and degraded by26S proteasome. The attached ubiquitin isremoved by the deubiquitinating enzyme (DUB) for recirculation. The function andmechanism of E2, E3and DUB has been wildly reported, but little systematic study ontheir regulatory network has been done.In this paper, kanamycin resistant gene was amplified by PCR and transferred intoSaccharomyces cerevisiae to knockout E2, E3and DUB genes respectively. The mutantstrains were screened by G418and confirmed by PCR. Ubiquitin binding domains aremodular domains that non-covalently bind to ubiquitin, which exist wildly in E2, E3andDUB. In this paper, a prokaryotic expression plasmid pGEX (kt)-4Q2was constructed byinserting artificially synthesized ubiquitin-binding domain gene Q2into pGEX (kt) vector.The plasmid was transferred into Escherichia coli BL21(DE3) to express a GST-fusionprotein by IPTG induction. The GST-fusion protein was purified by GSH resins.In this research,10E2null mutants,23E3null mutants and18DUB null mutantswere constructed as a foundation for the further investigation of their regulatory network.The GST-fusion protein was soluble expressed in Escherichia coli BL21(DE3) andsuccessfully purified by GSH resin, which could be used in the tandem affinity purificationof ubiquitinated proteins.