Expression And Purification Of TAT-PEⅢ Fusion Protein And Study Of Its Anti-tumor Activities

The malignant tumor seriously threatens health of mankind. Commonly cancer therapy treatments include surgery, radiotherapy and chemotherapy. However, the side effect of radiotherapy and chemotherapy is very severe, because the tumor cells and normal cells were simultaneously killed during treatment. Being a promising method, targeted biotherapy is a novel treatment for cancer therapy.Pseudomonas aeruginosa exotoxin A (Pseudomonas aeruginosa exotoxin A, PE) is a 66KD molecular weight single-chain protein. Through mediating the ADP-ribosylation of elongation factor -2, PE inhibits protein synthesis of target cells, by which leads to cell death finally. Comparing with anti-cancer chemicals,PE,as far as we know, is one of the most toxic molecules with much higher capability of killing cells,.Therefore, it is often used to build PE immunotoxin. PEâ…¢, a small part of the active region of PE and being with molecular weight of 25KD, has potential utilizable values to anti-cancer therapy. Although PEâ…¢has multiple prospects, it is limited to be applicated in clinical works due to its poor penetration.One way to overcome these flaws is the use of HIV TAT-mediated bacterial toxin. The HIV Tat protein transduction domain(PTD), a small basic cell-penetrating peptide(CPPs), has successfully been shown to deliver large variety of cargoes into cells, including peptides, proteins, and nucleic acids.. To reduce its toxicity to normal tissues, the TAT-mediated toxin can be directly injected into tumor tissues .Our previous works have demonstrated that the TAT-PE fusion protein has antitumour activities both in vitro and in vivo, and has the potential to be used for targeted cancer therapy. In the present work, the cytotoxic effect of TAT-PEâ…¢fusion protein was conducted futher. The main works include items as follows:1. Construction the pQE-EGFP vector and expression EGFP proteinIn our experiments, the full-length EGFP gene was cloned into pQE-31 vector, named pQE-EGFP. The resulting vector was then transformed into E. coli JM109 and its induction was carried out with IPTG. The expression of EGFP was identified by running SDS-PAGE and Western blotting. These results showed that the molecular weight of the expressed protein is 28.5 kD, and the expression rate is 25%.2. Expression of TAT-EGFP protein and analysis of transduction efficiencyA pair of primers were designed for amplification of TAT-EGFP gene by PCR using pQE-EGFP plasmid as template,. In this primer pair, the part 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 cloned into pQE-31 vector. The recombinant pQE-TAT-EGFP was transformed into E. coli JM109 and induced with addition of IPTG for its expression. The expression of TAT-EGFP was identified by SDS-PAGE and western blotting, then purified by Ni2+-metal chelate affinity chromatography. In order to observe the activity of transmembrane about TAT, purified TAT-EGFP and EGFP proteins were added into cultured B16 cells in vitro, respectively, and the transduction efficiency was detected using fluorescence assay. A significant increase of fluorescence intensity in TAT-EGFP group was observed compared with that of EGFP control.3. Construction the pQE-PEâ…¢vector and expression PEâ…¢proteinThe PEâ…¢gene fragment was inserted into the prokaryotic expression vector pQE-31. The resulting vector pQE-PEâ…¢was transformed into E. coli JM109 and induced with IPTG for protein expression. The expression of PEâ…¢was identified by SDS-PAGE. The data indicated that PEâ…¢protein was stably expressed in strain JM109.4. Purification and refolding of PEâ…¢, TAT-PEâ…¢fusion protein(1) The recombinant vectors, pQE-PEâ…¢ans pQE-TAT-PEâ…¢, nearly express their fusion protein in the inclusion-body. The inclusion body of fusion protein was extracted from cultures of stably transformed E. coli JM109, and then dissolved in 8 mol/L urea. (2) PEâ…¢and TAT-PEâ…¢fusion protein were purified by Ni-NTA affinity chromatography column, respectively. (3) The purified fusion proteins were renatured through dialysis. 5. The assay of cytotoxic effect of TAT-PEâ…¢and PEâ…¢in B16 cellsB16 cells were treated with TAT-PEâ…¢and PEâ…¢respectively. Apoptosis assay with the Annexin V/PI double staining method for flow cytometry analysis is applied to determine their anti-tumor activity of the PEâ…¢and TAT-PEâ…¢fusion protein in vitro. These results suggested that the cytotoxic effect of TAT-PEâ…¢fusion protein on B16 melanoma cell is higher than PEâ…¢.Based on this favourable profile, further research concerning the potential anti-tumor activity of TAT-PEâ…¢fusion protein should be conducted precisely.