RGD多肽与尿激酶原嵌合分子的构建及性质研究

Construction and Characterization of the Chimeric Protein Consisting RGD-containing Peptide and ProUK

利用定点突变及DNA重组技术 ,将编码RGD多肽 (GPRGDWRMLG)的双链DNA片段 ,定向插入到相对于编码尿激酶原的Gly118 Leu119的cDNA分子中 ,构建了尿激酶原的嵌合体基因 ,并在甲醇酵母表达系统中进行了分泌表达。经过Zn2 + 螯合柱及SP阳离子柱两步层析后 ,目的蛋白质被纯化。经纤维蛋白平板测活 ,嵌合分子的比活为 6 5 0 0 0IU mg。嵌合分子与尿激酶相比 ,对显色底物S2 44 4作用的催化效率偏低 ,但具有较强的抑制血小板聚集的功能 ,抑制常数IC5 0为 2 1μmol L。以上结果表明嵌合分子不但具有较强的溶栓功能 ,而且具有抗栓功能 ,很可能是一种具有发展前景的双功能溶栓

On the basis of structure analysis and computer modeling of proUK,two\|chain DNA fragment encoding RGD peptide was inserted into the corresponding proUK cDNA site between Gly118\|Leu119,using site\|directed mutagenesis and DNA recombinant techniques.The chimeric gene was expressed in methylotrophic yeast Pichia pastoris expression system.The chimeric protein was purified after two step purification of Zn 2+ chelating column and SP cation exchange column.The specific activity was 65,000 IU/mg protein.The chimeric protein had somewhat lower catalytic efficiency (kcat/Km) on the substrate S2444 as compared to Urokinase.But it had high anti\|platelet aggregation activity,and the half inhibit constant was 2.1μmol/L.The results showed that the chimeric protein not only had higher thrombolytic activity but also obtained anti\|thrombus function.Further evaluation of the thrombolytic and antithrombolytic potential in appropriate animal models seemed to be investigated.