Purification and characterization of recombinant human macrophage colony-stimulating factor expressed in Chinese hamster ovary cells.
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.
Takahashi
M
M
Cellular Technology Institute, Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan.
Nishida
T
T
Takano
M
M
Yamanishi
K
K
Shimokura
M
M
Ohmoto
Y
Y
Aihara
K
K
Ichikawa
H
H
Nakai
S
S
Hirai
Y
Y
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
D011994:Recombinant Proteins; D016173:Macrophage Colony-Stimulating Factor; D013762:Tetrahydrofolate Dehydrogenase