Protein Modification: Combined Application Of Site-Specific Mutagenesis And Pegylation, And Preliminary Functional Verification

Objective: To obtain a new site-specific pegylated protein by site-specific mutagenesis to Staphylokinase(S) protein gene sequence and progressing pegylation on mutant protein on variant circumstances. Then select the optimum modification condition, and use molecular sieve to obtain high purity of modified protein.Methods: Primer was designed according to the crystal structure and antigen sites of S protein, the selected amino acid on S protein was mutant into cysteine by PCR; Mutant plasmid was obtained and transformed into BL21(DE3) cell. Mutant protein S-cys was expressed in escherichia coli cells(E. coli) through classic prokaryotic expression technology. Purified mutant protein was obtained by using ion affinity chromatography, ion exchange chromatography and molecular sieve. Pegylation was progressed under variant circumstances, and the influence on modification rate of time, temperature and mole ratio was observed and evaluated, then optimum modification condition was screened. Higher purity of pegylated protein peg-S-cys was obtained by secondary separation through molecular sieve from unsuccessfully modified protein. Biological activity was. Preliminary validated by fibrin-agarose plate process and animal embolism model.Results:(1)Cystein on target site was obtained successfully by site-specific mutagenesis.(2)High purity of S-cys protein was obtained by prokaryotic expression and purification.(3)S-cys protein was modified in a variety of conditions, optimal modify condition was screened according to the result of SDS-PAGE.(4) High purity of peg-S-cys protein was obtained by molecular sieve.(5) Thrombolytic ability of peg-S-cys was confirmed preliminary.Conclusion: site-specific pegylated protein was successfully obtained by site-specific mutagenesis and pegylation, and the reconstructed protein was biologically active in vitro and vivo.