Construction, Expression, Purification And Characterization Of Novel Staphylokinae Variants With RGD Motif At Their N-terminus

Intravascular thrombosis was one of the main causes of a wide variety of cardiovascular diseases. The agents currently used in the clinics included recombinant tissue-type plasminogen activator (rt-PA), recombinant streptokinase (r-SK), urokinase (UK) and their variants. Although the thrombolytic agents effectively reduced the mortality of thromboembolic diseases, they had such shortcomings as incomplete recanalization, delayed reperfusion time, reocclusion, bleeding and allergic reaction etc.Staphylokinase (SAK) was an extracellular protein secreted by numerous strains of Staphylococcus aureus(S. aureus). SAK was a promising thrombolytic agent, which at least equipotent to rt-PA for acute myocardial infarction and significantly more fibrin-selective. Furthermore it was easy to be over-expressed in Escherichia coli. But clinical examination suggested that there were some probabilities of reinfarct after recanalization with administration of recombinative SAK , which mainly results from hematoblast aggregation.Thus, it was a kind of affirmative therapeutic strategy preventing reinfarct to undergo anti-thrombolytic and antiplatelet therapy coinstantaneous with fibrinolytic therapy. It was known that platelet aggregation played an important role in the development of thrombus and depended on the exposure of membrane glycoprotein IIb/IIIa(GPIIb/IIIa)on the platelet surface. Based on the tripeptide arginine-glycine-asparatic acid (RGD) motif, many GPIIb/IIIa antagonists were designed and developed. In order to develop novel thrombolytic agent with fibrinolytic activity and antiplatelet activity, four SAK variants were designed and constructed, in which RGD motif was inserted into the N-terminus of SAK.Site-directed mutation was used to obtain the mutative genes of SAK by PCR, with designed mutative primers including RGD tripeptide sequences in N-terminal and template of wild type SAK plasmid. Then the fragment of SAK mutant was inserted into expression plasmid pBV220 and recombinant vectors were transformed into E.coli BL21, which could be induced by increasing the culture temperature from 30 to 42°C. The expressed variants are subsequently purified employing a three-step chromatographic purification process and fibrinolytic and antipletlet aggregation activity of purified mutants were analyzed.SDS-PAGE analysis indicated that the SAK mutants occupied more than 50% of the total proteins in E.coli BL21. The SAK mutants were purified by a similar three-step chromatographic purification process and their ,purities were more than 97%. The Specific fibrinolytic activities of SAK mutants were 10.8×104, 10.1×104, 11.0×104 and 11.2×104 HU/mg, and anti-platelet aggregation activity were 10.72%, 12.36%, 19.71% and 21.65%, respectively. The result of ELISA demonstrated their residual quantities of bacterial proteins were <0.1%, which accorded with the standards in"Pharmacopoeia of the People's Republic of China (2005 Edition)".In this paper, SAK variants are successfully expressed, and purified. They had higher thrombolytic efficacy and antiplatelet aggregation activity. The high purity and bioactivities of staphylokinase variants lay a good basis for manufacture and clinical application.