Purification of Recombinant Glycosylated Human Gamma Interferon Expressed in Transformed Chinese Hamster Ovary Cells
- 1 January 1984
- journal article
- research article
- Published by Mary Ann Liebert Inc in Journal of Interferon Research
- Vol. 4 (4), 461-468
- https://doi.org/10.1089/jir.1984.4.461
Abstract
Human IFN-gamma was produced in cultures of a Chinese hamster ovary (CHO) cell line transformed with a combination of plasmids encoding HuIFN-gamma cDNA and mouse DHFR cDNA and subsequently selected for growth in the presence of methotrexate. Confluent monolayers of these cells constitutively secrete HuIFN-gamma into the medium reaching a concentration of 2-5 micrograms/ml; the supernatant of the monolayer could be harvested daily for a period of more than 10 days. IFN-gamma was purified by passing the filtered CHO cell culture medium directly through a phosphocellulose column followed by elution and adsorption on a Con A-Sepharose column. Further concentration on an AMICON PM 10 filter and removal of high mw contaminating proteins with DEAE-Sephacel resulted in a IFN-gamma preparation of more than 99% purity (specific activity of about 10(8) International units per mg of protein). Each liter of CHO conditioned culture medium yielded 1-2 mg pure HuIFN-gamma. Its molecular weight, as determined by gel filtration, is about 50 kD and corresponds to a dimer structure. SDS-polyacrylamide gel electrophoresis indicated the presence of a 21 kD and a 25 kD polypeptide as compared with 17 kD for unglycosylated, bacterially made HuIFN-gamma and consistent with the two glycosylated forms of HuIFN-gamma produced in mitogen-stimulated human lymphocyte cultures.Keywords
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