HIGH-LEVEL PRODUCTION OF HUMAN BLOOD-COAGULATION FACTOR-VII AND FACTOR-XI USING A NEW MAMMALIAN EXPRESSION VECTOR

Recombinant human proteins are generally recovered in low yields from mammalian tissue culture following transfection with commercially avai lable vectors. We have constructed a novel vector containing both the neomycin-resistance-encoding gene (neo) as a dominant selectable marke r, and the dihydrofolate reductase-encoding gene (DHFR) to enable ampl ification of transfected DNA followed by stable expression in mammalia n cell lines. Levels of 5 mu g/ml of the coagulation proteins, factor VII (FVII) and factor XI (FXI), have been achieved in serum-free media . N-terminal sequencing of the purified proteins, and of their separat ed chains after proteolytic activation, demonstrated correct processin g of the recombinant products. In addition, the ratios of clotting act ivity to antigen for each are close to unity, and the recombinant and plasma-derived proteins had identical mobilities upon electrophoresis in the presence of SDS. The vector described will be of use for the sy nthesis of recombinant proteins, both wild-type and variants produced by site-directed mutagenesis, especially where complex post-translatio nal modification of the protein makes it essential to use mammalian ce lls.