Production of a recombinant antithrombotic and fibrinolytic protein, PLATSAK, in Escherichia coli.
The three main components involved in thrombosis and haemostasis are thrombin, platelets, and plasmin. Almost all inhibitors of thrombosis are focused either on the inhibition of thrombin or on the inhibition of platelets. We designed a construct using the fibrinolytic activity of staphylokinase, fused via a cleavable linker to an antithrombotic peptide of 29 amino acids. The peptide was designed to include three inhibitory regions: (1) the Arg-Gly-Asp (RGD) 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 29 amino acid peptide was reverse translated, and the gene was chemically synthesised and cloned into an expression vector as a 3' fusion to the staphylokinase gene. Gene expression was induced in E. coli Top 10 cells and the fusion protein, designated PLATSAK, was purified using metal affinity chromatography. The purified fusion protein significantly lengthened the activated partial thromboplastin time and thrombin time and inhibited the amidolytic activity of thrombin. The fibrinolytic activity was almost equal to that of recombinant staphylokinase as measured with a thrombelastograph. Platelet aggregation was not markedly inhibited by PLATSAK, probably due to the unfavourable three dimensional structure, with the Arg-Gly-Asp sequence buried inside. Our results confirm that it is feasible to design and produce a hybrid multifunctional protein that targets various components of the haemostatic process.
van Zyl
W B
WB
Department of Haematology, University of the Orange Free State, Bloemfontein, South Africa.
Pretorius
G H
GH
Hartmann
M
M
Kotzé
H F
HF
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli; D005343:Fibrinolytic Agents; D005344:Fibrinopeptide A / Q000235:genetics / Q000378:metabolism; D005813:Genes, Synthetic; D006629:Hirudins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics
D005343:Fibrinolytic Agents; D006629:Hirudins; D009842:Oligopeptides; C112515:PLATSAK protein, recombinant; D011993:Recombinant Fusion Proteins; D005344:Fibrinopeptide A; C047981:arginyl-glycyl-aspartic acid; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus