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Antigen‐independent selection of stable intracellular single‐chain antibodies
Author(s) -
Auf der Maur Adrian,
Escher Dominik,
Barberis Alcide
Publication year - 2001
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(01)03101-5
Subject(s) - selectable marker , complementarity determining region , hypervariable region , transfection , intracellular , computational biology , antigen , antibody , chemistry , microbiology and biotechnology , gene , biology , biochemistry , immunoglobulin light chain , genetics , plasmid
The intracellular expression of single‐chain Fv antibody fragments (scFv) in eukaryotic cells has an enormous potential in functional genomics and therapeutics [Marasco (1997) Gene Ther. 4, 11–15; Richardson and Marasco (1995) Trends Biotechnol. 13, 306–310]. However, the application of these so‐called intrabodies is currently limited by their unpredictable behavior under the reducing conditions encountered inside eukaryotic cells, which can affect their stability and solubility properties [Wörn et al. (2000) J. Biol. Chem. 275, 2795–2803; Biocca et al. (1995) Bio/Technology 13, 1110–1115]. We present a novel system that enables selection of stable and soluble intrabody frameworks in vivo without the requirement or knowledge of antigens. This system is based on the expression of single‐chain antibodies fused to a selectable marker that can control gene expression and cell growth. Our results show that the activity of a selectable marker fused to well characterized scFvs [Wörn et al. (2000) J. Biol. Chem. 275, 2795–2803] correlates with the solubility and stability of the scFv moieties. This method provides a unique tool to identify stable and soluble scFv frameworks, which subsequently serve as acceptor backbones to construct intrabody complementarity determining region libraries by randomization of hypervariable loops.