Prokaryotic expression,purification and protein activity detection of human Delta-like4~(ext)-93-217

AIM:To express and purify the fusion protein TRX/hDll4ext-93-217 and to detect its protein activity.METHODS:The cDNA sequence of hDll4ext-93-217 was amplified by PCR and cloned into the vector pMD18T.After sequencing,the correct fragment was cloned into prokaryotic expression plasmid pET32a(+).The target protein was expressed in E.coli BL21 induced by IPTG and the expression was detected by SDS-PAGE and Western blotting.The activity of purified target protein with NI-NTA was detected by luciferase report assay.RESULTS:The prokaryotic expression vector pET32a(+)-hDll4ext-93-217 was successfully constructed.The expression of the target protein,as a soluble fusion protein,was detected by SDS-PAGE and Western blotting,and the fusion purified protein of 1.5 g/L was obtained.The activity of the fusion protein TRX/hDll4ext-93-217 to stimulate the reporter gene was significantly higher compared with control(P=0.0154) or TRX(P=0.0227)(P0.05).CONCLUSION:A soluble fusion protein TRX/hDll4ext-93-217 is successfully obtained,which can effectively activate the notch signaling pathway.