title: Purification and characterization of recombinant human macrophage colony-stimulating factor expressed in Chinese hamster ovary cells.
abstract: 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 80-90 kD M-CSF as a homodimer and a molecular form higher than 150 kD. 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 42 kD. These purified rhM-CSFs had substantially the same specific activity (1 to 4 x 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.
authors.affiliation: Cellular Technology Institute, Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Takahashi
authors.forename: T
authors.initials: T
authors.lastname: Nishida
authors.forename: M
authors.initials: M
authors.lastname: Takano
authors.forename: K
authors.initials: K
authors.lastname: Yamanishi
authors.forename: M
authors.initials: M
authors.lastname: Shimokura
authors.forename: Y
authors.initials: Y
authors.lastname: Ohmoto
authors.forename: K
authors.initials: K
authors.lastname: Aihara
authors.forename: H
authors.initials: H
authors.lastname: Ichikawa
authors.forename: S
authors.initials: S
authors.lastname: Nakai
authors.forename: Y
authors.initials: Y
authors.lastname: Hirai
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006801:Humans; D016173:Macrophage Colony-Stimulating Factor / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D014162:Transfection
chemical_list: D011994:Recombinant Proteins; D016173:Macrophage Colony-Stimulating Factor; D013762:Tetrahydrofolate Dehydrogenase