Expression of human antithrombin III (AT III) at high levels has been achieved in
Chinese hamster ovary (CH0) cells by cotransfection and subsequent coamplification
of the transfected sequences. Expression vectors containing the AT III cDNA gene and
a dihydrofolate reductase (DHFR) cDNA gene were transfected into CH0 DHFR-deficient
cells. About 20% of the DHFR+ transformants secreted recombinant human AT III into the medium. Stepwise selection
of the AT III producing DHFR+ -transformants in increasing concentrations of methotrexate generated cells which
had amplified the AT III géne. We determined the copy number of the AT III cDNA and
the relative amounts of AT III specific mRNA at different stages of the amplification
process. Transfected CH0 cell lines expressed elevated immunreactive levels of human
AT III.
AT III secreted from these cell lines had the same molecular weight (60 kDa), immunological
properties and biological activities as AT III obtained from human plasma. In vivo
data concerning the inhibition of glycosylation by different drugs suggest that recombinant
AT III from CHO cells is glycosylated according to a complex type pattern.
