Characterization of Bifunctional Chimeric Molecule of PRGDWR Containing Pro-Urokinase
- VernacularTitle:PRGDWR-尿激酶原双功能嵌合分子的性质研究
- Author:
Xin DANG
;
Jingxin YANG
;
Qiang RU
;
Binggen RU
- Publication Type:Journal Article
- Keywords:
PRGDWR peptide;
single chain urokinase-type plasminoge n activator;
thrombolytic ability;
platelet aggregation
- From:
Progress in Biochemistry and Biophysics
2001;28(2):203-209
- CountryChina
- Language:Chinese
-
Abstract:
In order to obtain the bifunctional chimeric molecule of single-chain urokinase-type plasminogen activator (scu-PA) which can inhibit platelet aggregation, PRGDWR peptide was inserted into the site between Gly 118 and Leu119 (called insertion mutant B, InB). The recombinant gene of InB was expressed by Pichia pastoris. The secreted protein was purified by metal chelate affinity and strong cation exchange chromatography. The amidolytic ability of mutant InB is 5 900 IU/mg, the kinetic constants is: KInB m,plg=56.8 μmol*L-1，kInBcat,plg=0.33 s- 1. The kinetic constants of plasminogen activation reaction is: KInB m,plg=0.397 μmol*L-1，kInBcat,plg=0.0164 s-1. Fibrin inhibit the catalytiv ability of InB during plasminogen activatio n, the influence factor is 0.463(means InB remain 46.3% of the catalytic abili ty when fibrin was involved in the reaction system). The mutant not only has alm ost the same catalytic ability as wild type scu-PA, but also has strong ability of anti-platelet aggregation(compared with scu-PA), IC50 of InB is 12.7 μmol*L-1.
