High-level production of human blood coagulation factors VII and XI using a new mammalian expression vector.
Recombinant human proteins are generally recovered in low yields from mammalian tissue culture following transfection with commercially available vectors. We have constructed a novel vector containing both the neomycin-resistance-encoding gene (neo) as a dominant selectable marker, and the dihydrofolate reductase-encoding gene (DHFR) to enable amplification of transfected DNA followed by stable expression in mammalian cell lines. Levels of 5 micrograms/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 separated chains after proteolytic activation, demonstrated correct processing of the recombinant products. In addition, the ratios of clotting activity 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 synthesis of recombinant proteins, both wild-type and variants produced by site-directed mutagenesis, especially where complex post-translational modification of the protein makes it essential to use mammalian cells.
Kemball-Cook
G
G
Haemostasis Research Group, Clinical Research Centre, Harrow, UK.
Garner
I
I
Imanaka
Y
Y
Nishimura
T
T
O'Brien
D P
DP
Tuddenham
E G
EG
McVey
J H
JH
D000818:Animals; D016466:CHO Cells / Q000187:drug effects; D006224:Cricetinae; D016895:Culture Media, Serum-Free; D004351:Drug Resistance / Q000235:genetics; D005167:Factor VII / Q000096:biosynthesis* / Q000235:genetics; D005172:Factor XI / Q000096:biosynthesis* / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D008727:Methotrexate / Q000494:pharmacology; D008969:Molecular Sequence Data; D009355:Neomycin; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics; D014162:Transfection / Q000235:genetics
D016895:Culture Media, Serum-Free; D011994:Recombinant Proteins; D005167:Factor VII; D005172:Factor XI; D013762:Tetrahydrofolate Dehydrogenase; D009355:Neomycin; D008727:Methotrexate