TAT-EGFP Fusion Protein's Expression, The Purification And The Identification Of Its Activity

Objective To construct a prokaryotic expression vector of TAT-EGFP fusion protein, induce its expression and purify expression products to study the TAT-EGFP fusion protein in human bladder cancer cells (EJ cells) in vitro role of the transmembrane fusion and the protein through the solid tumors organization by entering into the cell membrane to promote the role of As₂O₃, which makes the foundation for protein therapy for TAT-EGFP protein carrying biologically active substances of large molecules reach the tumor site.Methods PCR technology was used to amplify TAT-EGFP gene with a PEGFP-1 template and a couple of TAT sequence contained primes, the amplified genes were connected to pET30a vector into a recombinant pET30a-TAT-EGFP expression vector, then inducted the expresion of TAT-EGFP by IPTG. The induced cells was broken with super sound way to release cytoplasem.The collected supernatant liquid was purified by Ni-NTA His Resin Purify System. Add the fusion protein TAT-EGFP into the culture medium, and incubate together with the bladder cancer cells (EJ cells) and bladder cancer tissue, then observe with a fluorescence microscopy to detect fluorescence of bladder tumor cells. Deal with bladder cancer with arsenic trioxide (As₂O₃) and TAT-EGFP fusion protein, Observe cell growth inhibition and apoptosis conditions to explore if the TAT-EGFP fusion protein is helpful for the promotion As₂O₃ entering EJ cells.Results Sequencing results showed that sequence of amplified fragment consistent with the purpose of sequence,the pET30a-TAT-EGFP expression plasmids were successfully constructed. The purity of the TAT-EGFP proteins are about 90% with the concentrations over 1.24 mg / ml. After dealing with bladder cancer cells by TAT-EGFP fusion protein and arsenic trioxide (As₂O₃), the concentration of arsenic trioxide (As₂O₃) increased in cancers cells. and shows a dose-and time-dependent effect.Conclusion The recombinant expression plasmid pET30a-TAT-EGFP was constructed and expressed in E.coli successfully.The fusion protein after purifying through the membrane and entities has a significant role. TAT-EGFP fusion protein could enhance As₂O₃ accessing into human bladder cancer EJ cells and significantly promote the inhibitory activity of As₂O₃ on EJ cells.