Prokaryotic Expression And Purification For Canis MC4R Gene

ObjectiveTo construct recombinant prokaryotic expression vectors for expressing the protein of Canis MC4R and N-terminus. To purify the N-terminus protein of Canis MC4R to provide antigens for preparing polyclonal antibodies of Canis MC4R and establish good basis for further study in the structure and the biologic function of Canis MC4R.MethodsThe full coding sequenceof Canis MC4R and N-terminus were amplified by PCR and MC4R DNA was cloned into pMD18-T vector. The pMD18-T/c MC4R was identified by digesting with BamH I and Xho I, the target fragments were subcloned into PET-30a(+) vectors, which were named pET30a(+)/Canis MC4R and PET30a(+)/Canis N-MC4R, then they were transformed into E.coli transetta(DE3), in which the protein MC4R and N-MC4R were induced by IPTG. The two kinds of protein were detected by Western blot and SDS-PAGE. Then the fusion protein N-MC4R was purified by Ni-NAT resin.ResultsCanis MC4R was cloned into the pMD18-T vector and the prokaryotic expression Vector PET-30a(+) successfully, and the open reading frames for encoding Canis MC4R and N-terminus were correct. Canis MC4R protein and N-terminus protein were expressed successfully in E.coli transetta(DE3), the targeted protein band (Mr=37 kDa, 16 KDa) could be observed in SDS-PAGE as predicted, Western blot results demonstrated the two kinds of fusion protein could bind with the His antibody specifically. The N-terminus fusion protein was purified by Ni-NAT resin , whose purity was 95% above.ConclusionsThe prokaryotic expression plasmids PET30a(+)/Canis MC4R and PET30a(+)/Canis N-MC4R were constructed and expressed in E.coli transetta(DE3) successfully. SDS-PAGE and Western blot results showed that Canis MC4R, Canis N-terminus protein were obtained, Canis N-terminus protein has been puritified by Ni-NAT resin.