Preparation And Characterization Of Pegylation Staphylokinase

AIM The staphylokinase(SAK)gene sequence was mutated by the site-specific mutation technology,and the PEGylated SAK protein was progressed by expression,PEGylation,and purification consequently.The biological activity and immunogenicity of the PEGylated SAK had been measured.METHODS Primers was designed according to the crystal structure and antigen sites of SAK.The selected amino acid(No.84)was mutated into cysteine by PCR.The mutant gene sequence was transformed into BL21(DE3)cell and expressed with classic prokaryotic expression technology.The mutant SAK which had been expressed was purified by Ni-NTA sepharose.The higher purity PEGylated SAK was obtained by PEGylation and secondary separation.Soluble fibrin plate method and thromboelastogram(TEG)was used to characterize the fibrinolytic activity of this peg-SAK-cys and other five kinds of peg-SAK-cys proteins,which process by our research group with same methods.Besides,the immunogenicity of peg-SAK-cys was measured by Enzyme-linked immunosorbent assay(ELISA).The superior peg-SAK-cys was filtered out according to these functional research and tested by the animal embolism model,consequently.RESULTS The mutant plasmid was constructed successfully.The No.82 mutant SAK protein(peg-SAK-C82A)was expressed and purified well,and more than 90% purity of this protein was obtained by PEGylation and second purification.The fibrin plate assay revealed that the No.97 PEGylated SAK and No.104 PEGylated SAK(peg-SAK-C104G)were successfully reserved considerable fibrinolytic activity,similar results were observed in the thromboelastogram experiment.The enzyme-linked immune-sorbent assay revealed that the peg-SAK-C104 G possessed the lowest immunogenicity among these peg-SAK-cys proteins and the wild-type SAK.The peg-SAK-C104 G retained significant fibrinolytic activity in vivo experiment as well,according to the animal embolism model.CONCLUSION The SAK,with high fibrinolytic activity and low imunogenicity,can be successfully produced by site-specific mutation and PEGylation Technology.