Prokaryotic Expression, Polymerization And Binding With Benzimidazoles Of Tubulins From Echinococcus Granulosus

Objective:a-and β-tubulin isotypes from the parasitic tapeworm Echinococcus granulosus were artificially expressed in E.coli.We try to complete the binding study between benzimidazoles(BZs)and recombinant a-and β-tubulins in search of tubulin isotypes that plays an important role on the life process of E.granulosus and offers a target for new drugs.Methods:The expression level of tubulin isotypes in protoscoleces and cysts of E.Kgranulosus were analyzed by RT-PCR.EgTUB4 and EgTUA9 were expressed in prokaryotic expression system,purified using Nickel Affinity Chromatography,and identified by SDS-PAGE.The optical density change of microtubule solution which reflected the polymerization/depolymerization status was monitored by the turbidity assay for identifying the aggregation of recombinant tubulins.The ultrastructure of assembled tubulin was observed by Transmission Electron Microscopy.The effects of BZs on polymerization of recombinant tubulins were also assessed.After being threated with BZs,the expression levels of Tubulin-β(EgTUB)of protoscoleces were studied by RT-PCR and Western blot.Results:The result of RT-PCR showed that the expressions of Tubulin-as,Tubulin-a9,Tubulin-β2,Tubulin-β4 and Tubulin-β6 were higher than other tubulin isotypes in protoscoleces and cysts of E.granulosus.EgTUB4 and EgTUA9 were expressed,and rEgTUB4 and rEgTUA9 were capable of polymerizing into microtubule-like structures in vitro.When being incubated with tubulins,BZs had a little effect on polymerization of recombinant tubulins.RT-PCT and Western blot showed that BZs could effect the expression of EgTUB(EgTUB2,EgTUB4 and EgTUB6).Conclusion:First,two isotypes(EgTUB4 and EgTUA9)from the parasitic tapeworm E.granulosus were artificially expressed in E.coli and purified to obtain tubulins that were capable of polymerizing into microtubule-like structures in vitro.Second,The polymerization of rEgTUB4 and rEgTUA9 is insensitive to BZs,which give us a hint that EgTUB4 may not be the target of BZs,but it might play an important role in searching for new structures or new drugs.