Streptokinase is one of the best known thrombolytic agents with widespread clinical use. However, its use is not risk-free due to its immunogenicity, hemorrhagic complications and relatively short half-life in circulation. Specific PEGylation of cysteine residue is a useful technique for reducing most of these complications. The aim of this study was designing and producing a cysteine containing mutant of streptokinase, to be used for specific PEGylation. Glut-amic acid 263, which is a surface amino acid in the structure of streptokinase protein, was selected for replacement with cysteine amino acid by site directed mutagenesis. The Glu263 codon was changed to cysteine codon by SOEing PCR technique. Then, the intact and mutated streptokinase genes were inserted into expression vector pET-26b (+). The co-nstructs were transformed to Escherichia.coli Rosetta (DE3) strain and the proteins were expressed by IPTG induction. The proteins were confirmed by SDS-PAGE and western blot analysis, purified by Ni-NTA agarose affinity chroma-tography under denaturing condition with urea and Sephadex G-25 column was applied to remove urea to refold the pr-oteins. This study indicated that by using aforesaid vector and host, cysteine containing mutant gene is expressed well and it will be appropriate for specific PEGylation.