Expression of a fibrinogen fusion peptide in Escherichia coli: a model thrombin substrate for structure/function analysis.
The initial event in fibrin clot formation is the thrombin-catalyzed cleavage of the A alpha chain of human fibrinogen. Most of the information required for thrombin recognition and cleavage of the A alpha chain lies in the amino terminal 51 residue CNBr fragment. By selective modification of residues in this region, we probed the features that participate in thrombin interactions. We constructed a vector which expressed a tripartite protein (tribrid) consisting of amino acids 1 to 50 of the A alpha chain followed by 60 amino acids of chicken collagen and the beta-galactosidase protein from Escherichia coli. Cell lysates run on NaDodSO4-polyacrylamide gels contained the predicted band of molecular weight (mol wt) 125,000. The tribrid reacted with a monoclonal antibody, Mab-Y18, which recognizes the amino terminus of the A alpha chain. When cell lysates were incubated with thrombin, FPA was released. By including one heterogeneous oligonucleotide in the construction, we generated plasmids that encoded three specific amino acid substitutions. Surprisingly, changing Gly14 to Val did not alter thrombin cleavage, although recognition by Mab-Y18 was lost. Substitution of lie for Arg23 did not alter either thrombin cleavage or monoclonal recognition. Substitution of Leu for Arg 16 altered thrombin cleavage; unexpectedly, recognition by Mab-Y18 was not changed.
Lord
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Department of Pathology, University of North Carolina, Chapel Hill 27599-7525.
Fowlkes
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D000595:Amino Acid Sequence; D001483:Base Sequence; D001546:Bentonite; D015153:Blotting, Western; D004926:Escherichia coli / Q000235:genetics*; D005340:Fibrinogen / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D005344:Fibrinopeptide A / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013917:Thrombin
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D001546:Bentonite; D005344:Fibrinopeptide A; D005340:Fibrinogen; D013917:Thrombin