Expression, Purification And Identification Of Recombinant Tissue-Type Plasminogen Activator-RGDS Fusion Protein

Objective Human tissue plasminogen activator (tPA) belongs to the serine protease family. It converts plasminogen into plasmin and is used clinically to treat thrombosis. prokaryotic expression and purification of recombinant tissue plasminogen activator (tPA)-RGDS fusion protein, and preliminary identification of its biological activity.Methods 1)The fusion gene(tPA-RGDS) was obtained by polymera-se chain reaction (PCR) and then inserted into prokaryotic soluble pQE30 vector with His-tag to construct expression plasmid (pQE30-tPA-RGDS). pQE30-tPA-RGDS was expressed by E.coli BL21 under auto-induction and purified by Ni-NTA affinity chromatography column. the fibrinolytic activ-ity of His-tPA-RGDS in vitro was characterized by soluble fibrin plate met-hod.2)The fusion gene (3C-tPA-RGDS) was obtained by polymerase cha-in reaction (PCR) and prokaryotic expression conditions (vector, strains, temperature) were optimized. then inserted into prokaryotic soluble pW28 vector with His-tag to construct expression plasmid (pW28-3C-tPA-RGDS). pW28-3C-tPA-RGDS was expressed by E.coli Origami 2(DE3) under auto-induction and purified by Ni-NTA affinity chromatography column and DEAE anion-exchange chromatography column.3C protein was digested specificity PreScission protease to remove the His-tag. purifiled and analy-zed the state of aggregation in solution by Hiload Superdex 200 gel flittration column.The purity of the tPA-RGDS fusion protein was analyzed by SDS-PAGE and the identify of the fusion protein by Western blot. Finally,the fibrinolytic activity of tPA-RGDS in vitro was characterized by soluble fibrin plate method.Results PCR, restriction enzyme digestion and sequencing analysis demonstrated that the recombinant plasmid (pQE30-tPA-RGDS and pW28-3C-tPA-RGDS) were constructed successfully. The fusion protein (His-tPA-RGDS and His-3C-tPA-RGDS) were soluble expressed by E.coli under auto-induction. The fusion gene (3C-tPA-RGDS) was expressed at optimal levels in this condition prokaryotic expression system (pW28 carrier,Origami 2 strain and induction temperature of 37℃).The His-tag was removed by PreScission protease cleavage reaction. The tPA-RGDS fusion protein’s molecular weight is 70000 Da as shown by SDS-PAGE, which was removed the His-tag using PreScission protease cleavage reaction. The monomeric form of the fusion protein in solution as shown by SDS-PAGE. After purified by Ni-NTA column, DEAE chromatography column and Superdex 200 column, tPA-RGDS fusion protein of high purity was obtained from the cell supernatants. In vitro experiments showed that fibrinolytic activity of the recombinant tPA-RGDS was about 2.5×105 IU/mg.Conclusion This is the first report that soluble expressed by E.coli under auto-induction for recombinant tissue-type plasminogen activator. It is could provide valuable information for using prokaryotic auto-induction expression systems to produce tPA at industrial and pharmaceutical levels. The high purity and fibrinolytic activity for fusion protein were obtained successfully,which offers a base for the identification of targeting of the fusion protein.