[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.
Jiao
J W
JW
National Laboratory of Protein Engineering, College of Life Sciences, Peking University, Beijing 100871, China.
Ru
Q
Q
Yu
M M
MM
Ru
B G
BG
D000595:Amino Acid Sequence; D015153:Blotting, Western; D004274:DNA, Recombinant / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006801:Humans; D009842:Oligopeptides / Q000235:genetics*; D010843:Pichia / Q000235:genetics; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D011994:Recombinant Proteins / Q000235:genetics*; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics*
D004274:DNA, Recombinant; D009842:Oligopeptides; D010975:Platelet Aggregation Inhibitors; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C047981:arginyl-glycyl-aspartic acid; D014568:Urokinase-Type Plasminogen Activator; C067551:saruplase