Purification of Recombinant Glycosylated Human Gamma Interferon Expressed in Transformed Chinese Hamster Ovary Cells

Abstract

Human IFN-γ was produced in cultures of a Chinese hamster ovary (CHO) cell line transformed with a combination of plasmids encoding HuIFN-γ cDNA and mouse DHFR cDNA and subsequently selected for growth in the presence of methotrexate.⁽¹⁾ Confluent monolayers of these cells constitutively secrete HuIFN-γ into the medium reaching a concentration of 2-5 μg/ml; the supernatant of the monolayer could be harvested daily for a period of more than 10 days. IFN-γ 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-γ preparation of more than 99% purity (specific activity of about 10⁸ International units per mg of protein). Each liter of CHO conditioned culture medium yielded 1-2 mg pure HuIFN-γ. 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-γ and consistent with the two glycosylated forms of HuIFN-γ produced in mitogen-stimulated human lymphocyte cultures.