Novel recombinant thrombolytic and antithrombotic staphylokinase variants with an RGD motif at their N-termini

摘要

To develop a more potent thrombolytic agent, four Sak(staphylokinase) variants were constructed, in whichRGD (Arg-Gly-Asp) sequences are introduced intodiferent sites of the N-terminus of Sak. These variantswere successfully expressed in Escherichia coil DH5|áas soluble cytoplasmic proteins in a 5-litre fermentorand accounted for more than 40% of the total cellularprotein. The expressed proteins were subsequentlypurified, employing a similar three-step chromato-graphic purification process. SDS/PAGE and HPLC-MS analyses indicated that the purified proteins werealmost completely homogeneous, the purity of the vari-ants exceeding 95%. Further investigations into theproperties of the Sak variants showed that mutationsat the N-terminus significantly affected N-terminal me-thionine excision, and serine residues at the N-terminusof Sak appeared to play an important role in theprocess. Kinetic analysis of r-Sak (recombinant Sak) andits variants using plasminogen as substrate indicatedthat the mutations affected the proteolysis. In addition,a significant inhibitory effect of the Sak variants at2.0 |ìM was observed on the ADP-induced, aggregationof platelets compared with that of r-Sak, whether N-terminally cleaved or not (P < 0.05). Furthermore, theinhibitory activity of Sak variants after N-terminalproteolysis was higher than that of native Sak variants.