Cloning and expression of extracellular fragment of human DR5 in E.coli

Objective To clone the extracellular fragment of human DR5(death receptor 5)cDNA(eDR5)and express it in E.coli cells.Methods The eDR 5 was amplified from human liver cancer cell line HepG2 by RT-PCR(reverse tran scription PCR),and was cloned into the vector pM D19-T.A fter verified by sequencing,it was cut with restriction endonucleases BamHI and EcoRI,and then the eDR5 was extracted and inserted into the prokaryotic expression vector pGEX-4T -1 and verified by sequencing.Finally the recombinant expression plasmid pG EX-eDR5 was transformed into E.coli BL21 (DE3),and induced to express the GST-eDR5 fusion protein with IPTG.Expressed protein was identified by SDSPAGE.Results Sequence analysis proved that the eDR5 was correct and prokaryotic expression plasmid pGEX-eDR5 was constructed successfully.The GST-eDR5 fusion protein was expressed in E.coli BL21(DE3)at a high level which accounted for about 19% of the total bacterial cellular proteins.Conclusion The extracellular fragment of human DR5 cDNA was cloned successfully and was expressed at a high level in E.coli.This research laid a foundation for purifying human DR5 polypeptides and generatinganti-human DR5 poly clonal antibody.