Antigen‐independent selection of stable intracellular single‐chain antibodies

FEBS Letters

Barberis, Alcide¹  Escher, Dominik¹  Auf der Maur, Adrian¹ 

[1] ESBATech AG, Winterthurerstr. 190, CH-8057 Zurich, Switzerland

关键词: Intrabody;    Single-chain Fv antibody fragment;    Complementarity determining region;    Functional genomics;    Gene therapy;    Saccharomyces cerevisiae;   

DOI  :  10.1016/S0014-5793(01)03101-5

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown