Staphylokinase (Sak), an extracellular protein produced by certain strains of Staphylococcus aureus, is an activator of human plasminogen to plasmin, which can cause the lysis of fibrin clots. The gene sak of 411 bp encoded a muture protein (15 kDa). To develop a more efficient thrombolytic agent, Sak variant containing (Arg-Gly-Asp) RGD motif was constructed by site-directed mutagenesis with arginine in place of lysine (K35R) and aspartic acid in place of asparagines (N37D), and identified by DNA sequencing. RGD-Sak was over-expressed in Escherichia coli BL21 under the control of the strong promoter tac in the presence of IPTG as inducer. RGD-Sak was purified by Ni2+ -ProBond™ column with a specific activity of 68000 U/mg protein, a recovery yield of 46 percent and a purification factor of 3. This result provided the basis for further research of RGD-Sak.