Objective:To obtain a site-specific pegylated protein by site-specific mutagenesis to staphylokinase（SAK）protein gene sequence and to progres polyethylene glycol（PEG）on mutant protein on variant circumstances;to obtain modified protein with high purity by selecting the optimum modification condition and using molecular sieve and to identify its function in vivo and in vitro.Methods:Primer was designed according to the crystal structure and antigen sites of SAK protein;the selected amino acid on SAK protein was mutant into cysteine（Cys）by PCR. Mutant plasmid was obtained and transformed into BL21（DE3）cell. Mutant protein S-cys was expressed in escherichia coli cells through prokaryotic expression technology. Purified mutant protein was obtained. Pegylation was progressed under variant circumstances

and optimum modification condition was screened. Higher purity of pegylated protein peg-S-cys was obtained through molecular sieve. Biological activity was preliminary validated by fibrin-agarose plate process and animal embolism model. Results:Cys on target site was obtained successfully. High purity of S-cys protein was obtained. S-cys protein was modified

and optimal modify condition was screened. High purity of peg-S-cys protein was obtained. Thrombolytic ability of peg-S-cys was confirmed preliminary. Conclusion:Site-specific pegylated protein can be successfully obtained

and the reconstructed protein is biologically active in vitro and in vivo.