[Construction and characterization of the chimeric protein consisting RGD-containing peptide and proUK]

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 Zn2+ 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 2.1 mumol/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.