A focused antibody library for selecting scFvs expressed at high levels in the cytoplasm

We describe the construction and validation of a large synthetic human single chain antibody fragment library based on a unique framework and optimized for cytoplasmic expression. Focusing the library by mimicking the natural diversity of CDR3 loops ensured that the scFvs were fully human and functional. We show that the library is highly diverse and functional since it has been possible to isolate by phage-display several strong binders against the five proteins tested in this study, the Syk and Aurora-A protein kinases, the αβ tubulin dimer, the papillomavirus E6 protein and the core histones. Some of the selected scFvs are expressed at an exceptional high level in the bacterial cytoplasm, allowing the purification of 1 mg of active scFv from only 20 ml of culture. Finally, we show that after three rounds of selection against core histones, more than half of the selected scFvs were active when expressed in vivo in human cells since they were essentially localized in the nucleus.This new library is a promising tool not only for an easy and large-scale selection of functional intrabodies but also for the isolation of highly expressed scFvs that could be used in numerous biotechnological and therapeutic applications.Intrabodies are defined as antibody molecules which are ectopically expressed inside the cell [1,2]. The concept of using intrabodies can result in the induction of a phenotypic knockout either by directly inhibiting the function of the targeted antigen or by diverting a protein from its normal intracellular location [3]. The main advantage of using intrabodies instead of RNA inhibition is that the inhibition is done at the protein level. As such, it is possible to target post-translational modifications or a specific conformation of the antigen [4]. In addition, by targeting antibody molecules to specific subcellular compartments using addressing signals [5], the intrabody induced phenotypic knockout can be restrained to a specific cell compartment. Alto