Expression, Refolding And Bioactivity Research Of Extracellular Domain Of Human CTR

Calcitonin receptor (CTR) belongs to G protein coupled receptors (GPCRs) family groupB. The N-terminal（Nt）extracellular domain of calcitonin receptor is an important region forbinding to its polypeptide ligands.This experiment truncated the20-160amino acids in the N terminal of CTR, andsynthesized the corresponding gene optimized according to the preferred condons of E.coli,then the gene was inserted into pET32a(+), pETDuet and pET22b(+) vector to construct threedifferent recombinant plasmids. In addition, The gene of coding region corresponding to the22-140amino acid residues in the N-terminal extracellular domain was obtained by PCR withthe synthetic gene as a template, then the PCR product was inserted into pET22b(+) vector.The four different constructs were transformed into E.coli BL21(DE3) for expression. Theoptimal induction condition was explored by scanning the induction time, inductiontemperature and concentration of inducer. The results showed that all the target proteins wereexpressed in the inclusion bodies. Only the amount of inclusion bodies could be increased butno effect on improving the soluble expression of the target protein by the optimizing of theinduction condition. In order to acquire the soluble target protein, we explored a highefficiency refolding method. The refolding procedure was performed by dialysis, and thesoluble refolded protein was further purified by Ni-NTA column and HiTrap Q column.Finally, the protein was identified by Mass Spectrometry. An in vitro receptor-ligand bindingassay was carried out by Pull-Down technology. The binding activity of the target protein tothe salmon calcitonin (sCT) was verified. Among the four expression constructs,pET22b(+)-CTR140was the best construct with highest efficiency for refolding andpurification of the target protein. The refolded pET22b(+)-CTR140protein had a specificbinding activity with salmon calcitonin.Until now, there is no report about prokaryotic expression, refolding and purification ofthe human CTR worldwide. The method for in vitro activity assay of CTR was also rarelyreported. This study laid the foundation for the further research on the relationship betweenstructure and function of CTR. It also contributes to new drug development against CTRassociated diseases, particularly calcium-related disease.