Urokinase mutant with better fibrin-specificity.
A 150-156 amino acids-deleted single-chain urokinase-type plasminogen activator (dscu-PA) and its recombinant wild-type counterpart (rscu-PA) were both expressed in Escherichia coli. After denaturation and renaturation in vitro, the expressed products were both purified to a single silver-stained band by means of IgG affinity chromatography. After activation by plasmin, similar enzymatic constants based on the hydrolysis of synthetic substrate S2444 by the two-chain molecular forms of dscu-PA and rscu-PA, or native tcu-PA were observed, suggesting that no impairment had been exerted on the catalytic active site of dtcu-PA by the 150-156 amino acids deletion. In both in vitro fibrin-clot and 125I-fibrin sepharose lysis tests, dtcu-PA showed a significantly higher fibrinolytic activity than rtcu-PA or rscu-PA. Hardly any effect on the concentration of fibrinogen in plasma was found in dtcu-PA. It was concluded that dtcu-PA had a higher fibrin specificity and that tcu-PA could be provided with better fibrin specificity by means of mutation.
Department of Biochemistry, Nanjing University, China.
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Hua
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D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000378:metabolism; D005337:Fibrin; D008969:Molecular Sequence Data; D009154:Mutation; D010960:Plasminogen Activators / Q000096:biosynthesis / Q000235:genetics*; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014568:Urokinase-Type Plasminogen Activator / Q000096:biosynthesis / Q000235:genetics*
D011994:Recombinant Proteins; D005337:Fibrin; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator