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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 approx...
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Published in: | Bioscience, biotechnology, and biochemistry biotechnology, and biochemistry, 1993, Vol.57 (6), p.915-921 |
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Main Authors: | , , , , , , , , , , |
Format: | Article |
Language: | English |
Subjects: | |
Online Access: | Get full text |
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Summary: | 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 × 10
7
units/mg protein). Deglycosylation experiments with the latter rhM-CSF using chemical (trlftuoromethanesulfonic 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. |
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ISSN: | 0916-8451 1347-6947 |
DOI: | 10.1271/bbb.57.915 |