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  • Title: [Cloning expression and characterization of a multifunctional anticoagulated peptide gene in E. Coli].
    Author: Mu R, Qin Y, Cha Y, Jing Q.
    Journal: Zhonghua Yi Xue Za Zhi; 2002 May 10; 82(9):593-6. PubMed ID: 12133478.
    Abstract:
    OBJECTIVE: To investigate the feasibility about the cloning, expression and characterization of multifunctional anticoagulated peptide. METHODS: We designed a construct using glutathione S-transferase (GST) as a protein vector, fused via a cleavable linker to an antithrombotic peptide of 31 amino acids. The peptide was designed to include three inhibitory regions: (1) the Lys-Gly-Asp (KGD) amino acid sequence to prevent fibrinogen binding to platelets; (2) a part of fibrinopeptide A, an inhibitor of thrombin; and (3) the tail of hirudin, a potent direct antithrombin. The amino acid sequence of the 31 amino acid peptide was reverse translated, and the gene was chemically synthesized and cloned into an expression vector pGEX-5X-3 as a 3'fusion to the GST gene. Gene expression was induced in E. coli DH5 alpha cells and the fusion protein was purified using affinity chromatography. RESULTS: The purified fusion protein significantly lengthened the activated partial thromboplastin time (74.7 s tested in 80 micromol/L) and thrombin time (102.3 s tested in 80 micromol/L) and inhibited the amidolytic activity of thrombin 11% activity compared with the control tested in 100 micromol/L. The ADP-induced platelet aggregation was markedly inhibited by the purified fusion protein. The study has also shown that GST exhibits relevant activity of antiplatelet weaker than the purified fusion protein. 20.7% activity compared with the control tested in 100 micromol/L. CONCLUSION: Our results confirm that it is feasible to design a hybrid multifunctional protein that targets various components of the haemostatic process.
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