一种含精-甘-天冬氨酰三肽人纤溶酶原K区缺失突变体在巴斯德毕赤酵母中的表达、纯化与特性

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

摘要:

为获得具有抗血小板聚集作用的重组人纤溶酶原 (hPLG)，尝试了一种含有精-甘-天冬氨酰 (RGD) 三肽的hPLG K区缺失突变体 (RGD-hPLG-?K)。首先，从pDNR-LIB-HPLG中克隆出HPLG-?K。然后定点突变激活环内的Pro559为Asp559，形成RGD模序。构建的pPICZαA-RGD-HPLG-?K电转化巴斯德毕赤酵母GS115，甲醇诱导表达后可产生0.16 g/L培液的RGD-hPLG-?K，Ni-NTA层析后纯度可达90％以上；Western blotting证实所获RGD-hPLG-?K可与兔抗hPLG抗血清反应；其24 h尿激酶激活速率和纤溶活性与hPLG-?K无显著差别 (P=0.630，n=5)；经尿激酶激活后，RGD-hPLG-?K的血小板聚集抑制率 (21.8％±1.57％) 显著高于hPLG-?K (3.8％±0.33％) (P=0.000，n=5)。表明成功构建、表达了一种具有抗血小板聚集活性的hPLG突变体，为研究新型多功能溶栓药物奠定了基础。

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.