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We expressed human macrophage colony-stimulating factor (M-CSF) in Chinese hamster ovary (CHO) cells by introducing an expression plasmid coding for a 554-amino-acid M-CSF precursor and dihydrofolate reductase (DHFR) gene, and by amplifying the sequence. A cell line was obtained that secreted approximately 200,000 units/ml after 6 days in culture. The expressed recombinant human M-CSF (rhM-CSF) primarily consisted of two molecular species, a main 80-90kD M-CSF as a homodimer and a molecular form higher than 150kD. Purification of a main rhM-CSF gave an apparently homogeneous protein disulfide-bonded from 42-kD subunits, but one of the purified rhM-CSFs was composed of two subunit species with molecular masses of 44 and 42kD. These purified rhM-CSFs had substantially the same specific activity (1 to 4×10^7 units/mg protein). Deglycosylation experiments with the latter rhM-CSF using chemical (trifluoromethanesulfonic acid) and enzymatic methods found a terminal neuraminic acid in addition to N- and O-glycosylation, but the two subunit species did not coalesce into a single molecule.