Expression of the second epidermal growth factor-like domain of human factor VII in Escherichia coli.
The second epidermal growth factor (EGF)-like domain of human coagulation factor VII is a potent inhibitor of the FVIIa/tissue factor complex, the predominant initiator of coagulation in vivo. This domain has now for the first time been cloned and expressed in Escherichia coli as an affinity fusion protein. The fusion protein was secreted into the periplasm of E. coli and purified by affinity chromatography. The purified protein consisted of a fusion protein with the expected molecular weight, and in addition, a significant fraction of oligomers cross-linked by intermolecular disulfide bonds. Despite the presence of oligomers, the purified protein was a potent inhibitor of the extrinsic blood coagulation pathway with an IC50 value of about 20 microM. The biological activity was retained after liberation of the EGF domain by proteolytic cleavage.
Hellebust
H
H
Nycomed, Bioreg Research, Oslo, Norway.
Bergseth
S
S
Orning
L
L
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004815:Epidermal Growth Factor / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D005167:Factor VII / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology; D015942:Factor VIIa / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000235:genetics; D013381:Subtilisins / Q000737:chemistry / Q000378:metabolism
D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D004815:Epidermal Growth Factor; D005167:Factor VII; D013381:Subtilisins; D015942:Factor VIIa