Summary
Tissue factor (TF) is the high affinity transmembrane receptor and cofactor for cellular
initiation of the plasma coagulation protease cascades by factor VIIa. We describe
the synthesis of recombinant huTF by stably transfected CHO cell lines carrying integrated
huTF DNA, and the isolation of huTF glycoprotein with specific functional activity
equivalent to natural huTF, The expression vector (pCDM8), carrying the cytomegalovirus
promoter to drive transcription of a partial cDNA construct encoding the complete
huTF protein chain, was cotransfected with a plasmid containing the neomycin resistance
gene for selection. These clones were further selected for level of expression of
huTF protein. Optimal expression compatible with stability and cell growth was approximately
13.5 x 106 molecules per cell. To our knowledge, this is one of the highest levels of expression
described for a recombinant transmembrane receptor in mammalian cells. Recombinant
huTF protein was obtained by single-step immuno-affinity purification, and exhibits
heterogeneity due to N-linked glycosylation. The protein was indistinguishable from
natural huTF based on functional properties of the glycoprotein reconstituted in lipid
vesicles, and expression of conformational epitopes. Large scale production of recombinant
huTF is feasible to permit basic studies of protein structure as well as for design
of huTF thromboplastin reagents.
