Purification and characterization of a kringle-deficit mutant of human plasminogen with Arg-Gly-Asp tripeptide expressed in Pichia pastorsis

Abstract:

To obtain a recombinant human plasminogen (hPLG) with potential anti-platelet aggregation activity, we cloned the cDNA coding Pro544 to Asn791 of hPLG, a kringle-deficit derivative (hPLG-?K). The Pro559 in activation loop was then mutated into Asp559 to provide Arg-Gly-Asp (RGD) motif. The constructed pPICZαA-RGD-HPLG-?K plasmid was expressed in yeast Pichia pastoris GS115, which produced RGD-hPLG-?K about 0.160 g/L broth. After affinity chromatography, the purity of the recombinant protein reached above 90%. Western blotting test confirmed that it retained the immunological reaction capability as human PLG. Its urokinase activation rate in 24 hours and its fibrinolytic activity made no deference against native hPLG-?K (P=0.630, n=5). Importantly, after activation by urokinase, RGD-hPLG-?K showed a significantly higher platelet aggregation inhibition rate (Ri) (21.8%±1.57%) than hPLG-?K (3.8%±0.33%) (P=0.000, n=5). These results proved that we constructed an hPLG mutant with anti-platelet aggregation activity, which made a foundation for developing innovative thrombolytic drugs with multifunction.