Hyperproduction of human interferon gamma by rat cells maintained in low-serum medium using the fibronectin gene promoter.
An expression vector, pF1900M, which expresses a cloned gene at a high level in quiescent mammalian cells was constructed using the rat fibronectin (FN) promoter. Human interferon gamma (HuIFN-gamma) cDNA inserted downstream of the FN promoter in pF1900M was introduced into rat 3Y1 cells and several IFN-producing cell lines were established. These cells secreted a low level of IFN when they were growing but secreted at a high level after they had reached confluence. The level was further increased when the confluent cells were maintained in low-serum medium and a cell line, I7, produced 4 x 10(5) IU/ml of IFN, comparable to that produced by genetically engineered Escherichia coli in 2 days. The IFN-producing ability of I7 cells could be maintained by successive replacements of low-serum medium for at least 2 weeks. HuIFN-gamma secreted into the medium had a molecular weight range of 22,000 to 25,000, similar to that of IFN-gamma produced by human lymphocytes. The N-linked glycosylation of HuIFN-gamma seemed to occur properly, since treatment of the IFN with N-glycanase resulted in a reduction of molecular weight to 17,000, which corresponds to that calculated from the deduced amino acid sequence of HuIFN-gamma.
Nakajima
T
T
Nichirei Research Institute, Tokyo.
Tsunoda
S
S
Nakada
S
S
Nagata
S
S
Oda
K
K
D000818:Animals; D001769:Blood; D015152:Blotting, Northern; D015139:Blotting, Southern; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D005353:Fibronectins / Q000235:genetics*; D005822:Genetic Vectors; D006031:Glycosylation; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis*; D007700:Kinetics; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011916:Rats, Inbred F344; D014162:Transfection
D016895:Culture Media, Serum-Free; D005353:Fibronectins; D012333:RNA, Messenger; D007371:Interferon-gamma; D004247:DNA