期刊
JOURNAL OF PROTEOMICS
卷 73, 期 9, 页码 1777-1785出版社
ELSEVIER
DOI: 10.1016/j.jprot.2010.05.012
关键词
Affinity tag; Monoclonal antibody; Surface plasmon resonance; Phage display; Stable cell line; Mammalian cell expression
资金
- Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT)
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 l 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. (C) 2010 Elsevier B.V. All rights reserved.
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