Expression, purification and characterization of a novel staphylokinasewith anti-platelet aggregation activity

Abstract:

[Objective] To investigate construction and expression of novel staphylokinase (SAK) mutants with Arg-Gly-Asp (RGD) motif at its N-terminus, and to develop a novel thrombolytic agent with fibrinolytic activity and anti-platelet activity. [Methods] The fragments of SAK mutants were inserted into expression plasmid pBV220 and recombinant vectors were transformed into E. coli BL21, The SAK mutants could be expressed by rose of the culture temperature from 30°C to 42°C. After purified by a 3-step chromatography, their fibrinolytic and anti-platelet aggregation activity were analyzed. [Results] SDS-PAGE analysis indicated that the SAK mutants occupied 50% of the total proteins in E coli BL21. After purified by the 3-step chromatography, the purity of SAK mutants were more than 95%. The Specific fibrinolytic activities of SAK mutants purified were 10.8×104 and 11.0×104 HU·mg-1.Anti-platelet aggregation activity were 10.72% and 19.71%, which were significantly higher than that of wild-type SAK. Thus the SAK variants were successfully expressed, and purified. The high purity and bioactivities of staphylokinase variants lay a good basis for manufacture and clinical application.