title: [The construction and characterization of human pro-urokinase mutant].
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.
authors.affiliation: College of Animal Science and Technology, Northwest Sci-Tech University of Agriculture and Forestry, Yangling 712100, China.
authors.forename: Yong-Sheng
authors.initials: YS
authors.lastname: Yu
authors.forename: Yan-Rong
authors.initials: YR
authors.lastname: Zhou
authors.forename: Jian-Shen
authors.initials: JS
authors.lastname: Lu
authors.forename: Xiao-Jie
authors.initials: XJ
authors.lastname: Wu
authors.forename: Qing-Wang
authors.initials: QW
authors.lastname: Li
authors.forename: Ji-Xian
authors.initials: JX
authors.lastname: Deng
mesh_terms: 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*
chemical_list: D050505:Mutant Proteins; D011994:Recombinant Proteins; D014568:Urokinase-Type Plasminogen Activator; C067551:saruplase