Miscellaneous user applications
Construction of an antibody library
Antibody molecules, which are ectopically expressed inside the cell, are called intrabodies. They can be used to induce a phenotypic knockout, with the advantage of inhibiting at protein level rather than at RNA level, as in RNAi. The synthetic proteins scFv (single-chain variable fragment) can be such intrabodies, and Philibert et al. have used an optimized form, called scFv13R4, to construct a human scFv library.
18 spiked oligonucleotides, custom synthesized by IBA GmbH, were used to introduce degenerate CDR3 loops (complementarity determining regions, figure). After filtering steps to eliminate the nonexpressed clones, only the cytoplasmically expressed scFvs were retained in the library without compromising the diversity.
Philibert et al. conclude that this new library is a promising tool for facile and large-scale selection of functional intrabodies.
Figure 1: Library construction. Schematic outline of the steps followed during library construction. The critical steps are: introduction of tailored CDR3 loops in a unique human scFv framework; removal of non-expressed clones by fusion with the CAT enzyme and selection on CAM plates; recombination of the 13 VH and 5 VL libraries, and display on phage. VH, VL: the two scFv-domains (heavy and light chain). Reprinted with permission of BioMed Central.
Philibert, Audrey Stoessel, Wei Wang, Annie-Paule Sibler, Nicole Bec, Christian
Larroque, Jeffery G Saven, Jérôme Courtête, Etienne Weiss and Pierre Martineau (2007)
A focused antibody library for selecting scFvs expressed at high levels in the cytoplasm.
BMC Biotechnology 7:81
Open access at http://www.biomedcentral.com/1472-6750/7/81