Open AccessConstruction and characterization of trifunctional single‐chain urokinase‐type plasminogen activators
Author(s) -
Bi Qun,
Cen Xiaodong,
Huang Yixiu,
Zhu Shenggeng
Publication year - 2002
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.2002.02816.x
Subject(s) - hirudin , chemistry , linker , plasminogen activator , peptide sequence , urokinase , benzamidine , biochemistry , sepharose , affinity chromatography , antithrombin , escherichia coli , fusion protein , plasmin , peptide , microbiology and biotechnology , thrombin , biology , heparin , recombinant dna , gene , platelet , enzyme , genetics , computer science , immunology , endocrinology , operating system
Two chimeric proteins have been constructed. One consists of four parts: a portion of the low molecular mass single‐chain urokinase‐type plasminogen activator (scu‐PA‐32K, residues 144–411), a 15‐mer linker sequence, the C‐terminal amino‐acid sequence (residues 53–65) of hirudin (Hir), and an RGD sequence derived from the leech protein decorsin, i.e. scu‐PA(32 k)‐linker‐Hir (residues 53–65)‐RGD peptide. The other comprises two main segments: scu‐PA(32 k) and hirudin into which RGDSP is inserted between its residues 33 and 34, i.e. hirudin (residues 1–33)‐RGDSP‐hirudin (residues 34–65)‐scu‐PA(32 k). These two chimeric genes were expressed in Escherichia coli , and the products were purified by Zn 2+ ‐chelating Sepharose 4B chromatography and benzamidine Sepharose 6B chromatography. Our results suggested that these two chimeric proteins not only had plasminogen‐dependent fibrinolytic activity, but also possessed platelet aggregation inhibitory activity and antithrombin activity.