Research On High Level Expression, Purification And Activity Of Fusion Protein RPEA (Ⅰa+Ⅱ)-HphA

With the development of science and technology,Gene therapy has become more and more extensive in treating human cancers and other diseases.So the key factor for gene therapy is to find a safe and highly targeting efficient gene delivery carrier.Many gene delivey carrier have been used,But most of them have inherent limitations and disadvantages, for example,delivering genes passively and relying on uptaking into vesicular compartments by endocytosis.As a tool of gene threapy,gene delivery carrier have played a decisive role in its procedures.Some research have shown that gene transfection mediated by receptor and DEAE-dextran can enhance the endocytosis of cells and improve the transfer efficiency, but,all of them have not been applied at present.Pseudomonas aeruginosa exotoxin A(PEA)is a single chain protein.It has strong toxicity to all of the mammalian cells which have the PEA receptors in its surface.Because it has specific cell-binding, membrane-translocation and ADP-ribosylation functions,many scientists have restructed it to reutilize.HphA,which is encoded by gene PHS051 of Pyrococcus horikoshii OT3 strain and can combine with DNA stably,is a histone-like protein.Since HphA has rare codon in its genome,so the expression level is very low in E.coli.In order to get high level expression, we had synthesized the HphA genes which have the bias codon of E.coli.We have successfully fused the PEA(Ⅰa+Ⅱ)gene with the artificially HphA gene.Subsequently,we have constructed and expressed the fusion protein PEA(Ⅰa+Ⅱ)-HphA as a gene delivery carrier,which have the receptor-binding,membrane-translocation and DNA-binding functions.First,the four recombinant expression plasmids pET-20b-PEA (Ⅰa+Ⅱ)-HphA,pET-22b-PEA(Ⅰa+Ⅱ)-HphA,pET-28a-PEA(Ⅰa+Ⅱ) -HphA and pET-28a-His·Tag-thrombin-PEA(Ⅰa+Ⅱ)-HphA was constructed,which encode the fusion protein rPEA(Ⅰa+Ⅱ)-HphA.Then the four plasmid was transformed into the E.coli BL21(λDE3),BL21 (λDE3)Codon plus and BL21(λDE3)pLys S competent cells respectively. Through optimizing the expression condition,the fusion protein was highly expressed in three different kinds of inclusion bodies and the optimized expression is the plasmid pET-28a-PEA(Ⅰa+Ⅱ)-HphA in E.coli BL21 (λLDE3),the expression level could be up to 47%by surface scan.The immunoblotting assay has shown that this fusion protein rPEA(Ⅰa+Ⅱ) -HphA could combine with both mouse anti-HphA McAb and rabbit anti-LHRH-PE40 PcAb specificity.By fermentation we have got 11g/L engineering bacteria cells.The purity of fusion protein rPEA(Ⅰa+Ⅱ) -HphA inclusion bodies could achieve to 82%through clearaging by sonication and washing by 2 mol/L urea.After we had renatured and purified the fusion protein rPEA(Ⅰa+Ⅱ)-HphA inclusion bodies by different refolding method,the purity and renaturation rate were detected. Then the methods of renaturation and purification were optimized.That is first,refolding by dialysis;second,purified by Cu chelating chromatograph;third,desalinated by dialysis.Eventually,the renaturation rate could be reached to 10%and the concentration could be 0.64mg/ml. EMSA had shown that the fusion protein rPEA(Ⅰa+Ⅱ)-HphA could increase the mobility of linear plasmid.So we choose the pEGFP-C1 as report gene and the Hela as the target cell.After incubating for 48 hours, the rPEA(Ⅰa+Ⅱ)-HphA-pEGFP compounds were transfected Hela cells, then the green fluorescent had been shown under the fluorescent microscope.The compounds increased the transfection rates markedly compared with polylysine methods.Above all,the recombinant expression plasmids had been constructed,the fusion protein had expressed at high level,the fusion protein inclusion bodies had been successfully renatured.The fusion protein could succesfully transfer the EGFP gene into Hela cell and EGFP gene could be expressed.The results shown that the fusion protein can be serve as the target gene delivery carrier.In additional,the fusion protein can be change the receptor domain to suit for delivering therapeutic gene to different target cells or organizations.So,we belived that the fusion protein rPEA(Ⅰa+Ⅱ)-HphA have the potential to be applicated in clinical gene therapy as a gene delivery carrier.