Prokaryotic expression, purification and protein activity detection of human Delta-like4ext-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) (P ＜ 0.05). CONCLUSION: A soluble fusion protein TRX/hDll4ext-93-217 is successfully obtained, which can effectively activate the notch signaling pathway.
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