To achieve high-level production of human lymphotoxin (hLT), a plasmid (p beta LT-ldhfr) containing the hLT genomic DNA, a mouse dihydrofolate reductase (DHFR) cDNA, and a bacterial Ecogpt gene was cotransfected with a plasmid (p beta LTML) encoding only the hLT genomic DNA into Chinese hamster ovary (CHO-K1) cells at a 1:7 molar ratio. Subsequently one of the Ecogpt-positive clones (clone A31) was grown in stepwise increasing concentrations of methotrexate (MTX). A large amount of the hLT was secreted by cells resistant to increased levels of MTX as a result of coamplification of the DHFR cDNA and the hLT gene. A cell clone (clone M-1) resistant to up to 500 nM MTX constitutively expressed the hLT at a concentration of 80 micrograms per ml at an elevated level for about 2 months. The hLT was produced in glycosylated form the molecular mass of which was 23,000 daltons and the mRNA was normally spliced, so the protein molecules were probably homogeneous.
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