An antigen-mediated selection system for mammalian cells that produce glycosylated single-chain Fv.
Selection and production of specific antibodies are limiting the development of high-throughput immunoassays such as antibody chips. In this study, we propose an antigen-mediated selection of antibody producers (ASAP) system in mammalian cells. As a model system, transgenes encoding anti-fluorescein ScFv fused to cytokine receptors were introduced to IL-3-dependent cell lines. Addition of fluorescein-conjugated BSA induced growth signal through the ScFv/receptor chimeras, leading to selective expansion of the transduced cells. Cre recombinase was then used to excise the receptor gene flanked by two loxP recognition sites in the introns, resulting in secretion of his-myc-tagged ScFv to the culture medium. When the first loxP site was used in the exon as a linker between ScFv and receptor, enhanced antigen-mediated cell proliferation and production of unexpectedly glycosylated ScFv were achieved. ASAP is the first mammalian selection/production system of recombinant human ScFvs, without need for subcloning and with the advantage of glycosylated product.
Pihkala
Päivi
P
Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Kawahara
Masahiro
M
Ueda
Hiroshi
H
Nagamune
Teruyuki
T
D000818:Animals; D000941:Antigens / Q000276:immunology; D002460:Cell Line; D006031:Glycosylation; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D008024:Ligands; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D011995:Recombination, Genetic; D019076:Transgenes
D000941:Antigens; D007135:Immunoglobulin Variable Region; D008024:Ligands; D011993:Recombinant Fusion Proteins