[Cloning expression and characterization of a multifunctional anticoagulated peptide gene in E. Coli].
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.


CONCLUSIONS
Our results confirm that it is feasible to design a hybrid multifunctional protein that targets various components of the haemostatic process.
Department of Cardiovasology, Affiliated Changhai Hospital of Second Military Medical College, Shanghai 200433, China.
Ruibin
R
Mu
Yongwen
Y
Qin
Yanping
Y
Cha
Qing
Q
Jing
D000925:Anticoagulants / Q000378:metabolism / Q000494:pharmacology; D000991:Antithrombins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D006629:Hirudins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D006801:Humans; D010455:Peptides / Q000235:genetics / Q000494:pharmacology; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000378:metabolism / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology
D000925:Anticoagulants; D000991:Antithrombins; D006629:Hirudins; D010455:Peptides; D010975:Platelet Aggregation Inhibitors; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase