Expression in Escherichia coli of the human fibrinogen B beta chain and its cleavage by thrombin.
The human fibrinogen B beta chain was expressed in Escherichia coli to study the functions of fibrinogen associated with this subunit. Recombinant B beta chains were expressed at 100 ng/mL in an IPTG-dependent manner. A first cistron sequence, inserted into the expression vector 5' to the B beta chain cDNA, was required to express the protein. Recombinant B beta chains were expressed within five minutes after induction with IPTG and were soluble in physiologic buffers. The recombinant B beta chains migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at a rate identical to B beta chains from fibrinogen treated with N-glycanase. Recombinant B beta chains were cleaved by thrombin, as demonstrated by the loss of cross-reactivity with a monoclonal antibody (MoAb) specific for the undigested B beta 1-42 fragment. The levels of expression of the B beta chain were much lower than those reported previously for the gamma chain of fibrinogen expressed in a similar vector in E coli. However, these levels are sufficient to allow further characterization of this fibrinogen subunit.
Bolyard
M G
MG
Department of Pathology, University of North Carolina, Chapel Hill 27599-7525.
Lord
S T
ST
D001483:Base Sequence; D015153:Blotting, Western; D004274:DNA, Recombinant / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005340:Fibrinogen / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013917:Thrombin
D004274:DNA, Recombinant; D011994:Recombinant Proteins; D005340:Fibrinogen; D013917:Thrombin