title: A rapid screening method for cell lines producing singly-tagged recombinant proteins using the "TARGET tag" system.
abstract: Recombinant production of extracellular glycoproteins in stable mammalian cell lines is an ideal technique for obtaining a large quantity of high-quality proteins. In most cases, however, current methodologies do not allow for sufficiently rapid cell line development and protein purification. Here, we describe a 21-residue peptide tag (designated as TARGET tag) and its use for rapid stable cell line development and purification. The ability of the anti-tag antibody P20.1 to withstand repetitive regeneration cycles has enabled the development of a sensitive surface plasmon resonance-based screening format that requires only 20 microl of cell culture supernatants. Direct and semi-quantitative screening at the 96-well culture scale eliminated the need for a second screening, re-cloning, or sorting, thereby minimizing culture pre-production time. Using this system, "high producer" cell lines were established in less than a month, and milligram quantities of target proteins could be purified with a standardized protocol.
authors.affiliation: Laboratory of Protein Synthesis and Expression, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Sanae
authors.initials: S
authors.lastname: Tabata
authors.forename: Maiko
authors.initials: M
authors.lastname: Nampo
authors.forename: Emiko
authors.initials: E
authors.lastname: Mihara
authors.forename: Keiko
authors.initials: K
authors.lastname: Tamura-Kawakami
authors.forename: Ikuo
authors.initials: I
authors.lastname: Fujii
authors.forename: Junichi
authors.initials: J
authors.lastname: Takagi
mesh_terms: D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods; D006801:Humans; D019151:Peptide Library; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification*; D020349:Surface Plasmon Resonance / Q000379:methods; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins