Construction and expression of extracellular domain of human death receptor 5 and detection of its biological activity / 中国肿瘤生物治疗杂志

ABSTRACT

Objective: To construct the expressing vector of the extracellular domain of death receptor 5 (DR5), express it E.coli, identify the purified DR5 protein, and study its biological activity. Methods: The extracellular domain of DR5(eDR5) was assembled by overlapping PCR. The expression vector pET-22b(+)/ DR5 was constructed and transformed into E.coli BL21(DE3). The expression of eDR5 protein was induced by IPTG and purified by Ni 2+ -affinity chromatographic column. The purity and specificities were detected by SDS-PAGE and ELISA, respectively. The blocking effects of purified eDR5 on FMU1.5-induced apoptosis of U343, U373 cells were observed. Results: The extracellular domain of DR5 was obtained by overlapping PCR. The eDR5 protein was expressed in both supernatants and inclusion bodies with a yield more than 30% of total bacterial proteins. The purity of eDR5 was more than 95% and the yield reached 9 mg/ml. The result of ELISA showed the purified protein was eDR5. Purified eDR5 partially blocked the apoptosis of U343 cells induced by FMU1.5 and TRAIL. Conclusion: The successful construction, expression, and purification of the extracellular domain of DR5 protein lays a foundation for further study of DR5 function.