Expression and purification of recombinant, glycosylated human interferon alpha 2b in murine myeloma NSo cells.
We have expressed recombinant human interferon-alpha 2b in mammalian cells and isolated cell lines constitutively secreting very high levels of biologically active protein. The expression system takes advantage of the strong human cytomegalovirus immediate early promoter in mouse myeloma NSo cells and glutamine synthetase as a selectable marker; spontaneous mutants with amplified gene copy numbers were selected by growth of primary transfectants in the presence of methionine sulfoximine. Using this procedure, we have isolated a recombinant NSo cell line which secretes human interferon at the rate of 20 micrograms/10(6) cells/24 h and accumulates up to 120 micrograms/ml (approximately 2.4 x 10(7) U/ml) following prolonged undiluted culture. The interferon (IFN) could be efficiently purified on a polyclonal bovine anti-human IFN alpha specific antibody column and the glycosylation pattern was found to be similar to that of nonrecombinant IFN alpha 2b purified from virus-induced human Namalwa cells. The biological activity of the recombinant material was indistinguishable from that of natural IFN from Namalwa cells, and the specific antiviral activity, as assayed on human HeLa cells challenged with encephalomyocarditis virus, was 2 x 10(8) IU/mg, similar to that of nonrecombinant IFN preparations. This represents the highest reported level of glycosylated, recombinant IFN expression in a stable mammalian system and is a significant advance in the large-scale production of these clinically important cytokines.
Rossmann
C
C
Biology Division, Wellcome Foundation Ltd., Beckenham, Kent, England.
Sharp
N
N
Allen
G
G
Gewert
D
D
D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015703:Antigens, CD / Q000096:biosynthesis; D000943:Antigens, Differentiation / Q000096:biosynthesis; D002417:Cattle; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004686:Encephalomyocarditis virus / Q000737:chemistry / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D015870:Gene Expression; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D008562:Membrane Glycoproteins; D051379:Mice; D009101:Multiple Myeloma / Q000235:genetics / Q000378:metabolism*; D009699:N-Glycosyl Hydrolases / Q000096:biosynthesis; D010446:Peptide Fragments / Q000032:analysis; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins; D013997:Time Factors; D014162:Transfection / Q000235:genetics; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D000943:Antigens, Differentiation; D017931:DNA Primers; D007106:Immune Sera; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D009699:N-Glycosyl Hydrolases; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; C498155:Cd38 protein, mouse; D051997:ADP-ribosyl Cyclase 1