Antigen-independent selection of stable intracellular single-chain antibodies.
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.
Auf der Maur
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ESBATech AG, Winterthurerstr. 190, CH-8057, Zurich, Switzerland.
Escher
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Barberis
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D000937:Antigen-Antibody Reactions; D000941:Antigens; D002457:Cell Extracts; D002460:Cell Line; D022801:Complementarity Determining Regions / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D004268:DNA-Binding Proteins; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D017930:Genes, Reporter; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D016350:Leucine Zippers; D056892:Mediator Complex; D019151:Peptide Library; D011494:Protein Kinases / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000276:immunology; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D012995:Solubility; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism; D015533:Transcriptional Activation; D001616:beta-Galactosidase / Q000378:metabolism
D000941:Antigens; D002457:Cell Extracts; D022801:Complementarity Determining Regions; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C058337:GAL11 protein, S cerevisiae; C052130:GAL4 protein, S cerevisiae; D007135:Immunoglobulin Variable Region; D056892:Mediator Complex; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D011494:Protein Kinases; D001616:beta-Galactosidase