Selection of highly productive mammalian cells based on an inducible growth advantage using an antibody/receptor chimera.
In mammalian cell culture, the selection of high producers is a critical step in efficient recombinant protein production. Drug-resistance selection has been commonly used, but does not always give a pure population of high producers. In this study, we propose a novel selection method in which the growth of high producers is specifically promoted. Two plasmids encoding (i) a hybrid receptor composed of the V(H) portion of anti-hen egg lysozyme antibody HyHEL-10 and an N-terminally truncated erythropoietin receptor (V(H)-EpoR), and (ii) a V(L)-EpoR fusion derived from the same construct as in (i), were employed. The second plasmid contained enhanced green fluorescent protein (EGFP) as a model recombinant protein that was flanked by the internal ribosomal entry sequence. Both plasmids were used simultaneously to transfect an IL-3-dependent murine myeloid cell line, 32D. The transfectants, after antigen selection in the absence of IL-3, showed a clear antigen-induced dose-dependent proliferation. In addition, a high EGFP expression level was observed by flow cytometry in comparison with the cells before antigen selection. The results clearly demonstrate the advantage of our method over conventional drug-resistance selection. We propose the term AMEGA (Antigen MEdiated Genetically-modified cell Amplification) for such an approach.
Kawahara
Masahiro
M
Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-8656, Japan. kawahara@bio.t.u-tokyo.ac.jp
Ueda
Hiroshi
H
Tsumoto
Kouhei
K
Kumagai
Izumi
I
Mahoney
Walt
W
Nagamune
Teruyuki
T