[The construction and characterization of human pro-urokinase mutant].
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.
Yu
Yong-Sheng
YS
College of Animal Science and Technology, Northwest Sci-Tech University of Agriculture and Forestry, Yangling 712100, China.
Zhou
Yan-Rong
YR
Lu
Jian-Shen
JS
Wu
Xiao-Jie
XJ
Li
Qing-Wang
QW
Deng
Ji-Xian
JX
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000096:biosynthesis / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D014568:Urokinase-Type Plasminogen Activator / Q000096:biosynthesis / Q000235:genetics*
D050505:Mutant Proteins; D011994:Recombinant Proteins; D014568:Urokinase-Type Plasminogen Activator; C067551:saruplase