Expression and purification of TAT-EGFP fusion protein and study of its penetrating activity

Objective To purify the TAT-EGFP fusion protein and investigate its transduction efficiency. Methods By using pQE-EGFP plasmid as template,a pair of primers were designed for amplification of TAT-EGFP gene by PCR. In this primer pair,the sequence encoding the 11-amino-acid HIV TAT domain and flanking glycine residues were added to the 5'terminal of the forward primer. The TAT-EGFP PCR products were purified,and then cloned into pQE-31 vector. The recombinant pQE-TAT-EGFP was transformed into E.coli JM109 and induced with IPTG for protein expression. The expression of TAT-EGFP was identified by SDS-PAGE and Western blotting,and then purified by Ni2+-metal chelate affinity chromatography. The purified fusion protein was added into cultured B16 cells in vitro and the transduction efficiency was detected using fluorescence assay. Results The recombinant plasmid pQE-TAT-EGFP was constructed successfully. The results of SDS-PAGE and Western blotting showed that the molecular weight of the expressed TAT-EGFP fusion protein was 30 160. After purification,the TAT-EGFP fusion protein could cross the cytomembrane of B16 cells in vitro. Conclusion This experiment demonstrates the possibility of TAT to directly deliver protein into cells,which provides a basis for the research on the antitumor activity of TAT-PEⅢ fusion protein.