Production of recombinant granulocyte colony-stimulating factor by knocking into the active immunoglobulin heavy chain gene locus in the hybridoma cell line |
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Authors: | Yoshihisa Kuwana Kikuko Funayama Hiromasa Miyaji Mamoru Hasegawa Hajime Yoshida Seiga Itoh |
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Institution: | (1) Tokyo Research Laboratories, Kyowa Hakko Kogyo, Co. Ltd., 3-6-6 Asahi-machi, 194-8533 Tokyo, Japan |
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Abstract: | The hybridoma cell line KM50 originally produces a monoclonal antibody at a concentration of ∼40 mg ml-1 in ascites. To investigate the possibility to apply this expression system to the production of useful proteins, the cDNA
encoding human granulocyte colony-stimulating factor was inserted by homologous recombination into just downstream of the
promoter of the active immunoglobulin heavy chain gene of KM50. Site directed integration of targeting DNAs resulted in the
disruption of expression of the immunoglobulin heavy chain proteins with a frequency of 1 in 10 ∼ 100 G418-resistance transfectants.
One of the monoclonal antibody-deficient transfectants produced25 ng ml-1 of granulocyte colony-stimulating factor in the supernatant of its cell culture the number of molecules of which corresponds
to that of the monoclonal antibody originally produced by KM50. However, when this transfectant was injected intraperitoneally,
it produced only a 9 μg ml-1 concentration of granulocyte colony-stimulating factor in ascites, which is approximately 3 orders of magnitude less than
the monoclonal antibody. This method may be applicable to production of other recombinant proteins, although further optimization
in the conditions of production would be needed in order to reach much higher yields.
This revised version was published online in August 2006 with corrections to the Cover Date. |
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Keywords: | Granulocyte colony-stimulating factor (G-CSF) Homologous recombination Immunoglobulin Production Promoter Recombinant proteins |
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