The construction and characterization of human pro-urokinase mutant.
- Author:
Yong-Sheng YU
1
;
Yan-Rong ZHOU
;
Jian-Shen LU
;
Xiao-Jie WU
;
Qing-Wang LI
;
Ji-Xian DENG
Author Information
1. College of Animal Science and Technology, Northwest Sci-Tech University of Agriculture and Forestry, Yangling 712100, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Base Sequence;
Blotting, Western;
CHO Cells;
Cloning, Molecular;
Cricetinae;
Cricetulus;
Electrophoresis, Polyacrylamide Gel;
Humans;
Molecular Sequence Data;
Mutant Proteins;
biosynthesis;
genetics;
Recombinant Proteins;
biosynthesis;
genetics;
Transfection;
Urokinase-Type Plasminogen Activator;
biosynthesis;
genetics
- From:Chinese Journal of Biotechnology
2005;21(4):573-578
- CountryChina
- Language:Chinese
-
Abstract:
It is very easy for the pro-UK to lose it's biological activity because of the digestion of pro-UK by the thrombin or the inhibition of pro-UK by the PAI-I. So three pro-UK mutant (pro-UK) genes were constructed in this experiment with the PCR point-mutant method. The thrombin cleavage site Arg156 in pro-UK was mutated into His156, and named as pro-UKM1; PAI binding sites Arg178, Arg179, Arg181 were mutated into Lys178, Lys179, His181, named as pro-UKM2; The mutant containing His156, Lys178, Lys179, His181 as pro-UKM3. Three mutants were expressed in CHO cells respectively and analyzed with SDS-PAGE fibrin plate assay and western blot. The results showed that the three mutants and the native pro-UK have the same single electrophoresis band indicating most of the pro-UK was single chain. In vitro plasma clot lysis assays indicated that the pro-UKM1 have the ability to resistant against thrombin digestion; pro-UK2 could resist against PAI inhibition; while pro-UK3 improved resistances against both thrombin and PAI. It looks very promising that the pro-UK3 can be a new medicine of dissolving thrombus.
