Biosynthesis and biological evaluation of a neotype neurotrophin TAT-BDNF

Objective: To synthesis a neotype neurotrophin TAT-BDNF and identify its biological activity, for further investigation of an alternative for treatment of central nervous system injury with functional protein. Methods:With molecular cloning technique, The recombinant vector termed pTAT-HA-BDNF was constructed, which could encode both TAT protein transduction domain and human brain-derived neurotrophic factor without signal peptide. Purified TAT-BDNF fusion protein were obtained by prokaryotic expression, purification and renaturation. Then it was added into cerebellar granule cells cultured in vitro, while Immunofluorescence analysis of the protein transduction effect of TAT-BDNF and observation of neuroprotective effect from glutamate-mediated excitotoxicity insulted with Hoechst33342 staining were performed. Results: The purified fusion proteins by prokaryotic expression were confirmed by SDS-PAGE. Double Immunofluorescence assay showed that TAT-BDNF can rapidly be deliveried into cerebellum granular cell cultured in vitro. During the study of excitotoxic insult induced by glutamate, TAT-BDNF can decrease the apoptosis ratio and enhance neurons survival. Conclusions: TAT-BDNF fusion proteins produced by prokaryotic expression system possess biological effect of protein transduction and neuroprotection