Expression of a fibrinogen fusion peptide in Escherichia coli: a model
thrombin substrate for structure/function analysis
ST Lord and DM Fowlkes
Department of Pathology, University of North Carolina, Chapel Hill
27599-7525.
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.
Volume 73,
Issue 1,
pp. 166-171,
01/01/1989
Copyright © 1989 by The American Society of Hematology
