Isolation of antigen-binding camelid heavy chain antibody fragments (nanobodies) from an immune library displayed on the surface of Pichia pastoris.

TitleIsolation of antigen-binding camelid heavy chain antibody fragments (nanobodies) from an immune library displayed on the surface of Pichia pastoris.
Publication TypeJournal Article
Year of Publication2010
AuthorsRyckaert, S., E. Pardon, J. Steyaert, and N. Callewaert
JournalJ Biotechnol
Volume145
Issue2
Pagination93-8
Date Published2010 Jan 15
ISSN1873-4863
KeywordsAnimals, Antibodies, Monoclonal, Camelids, New World, Cell Membrane, Cloning, Molecular, Immunoglobulin Heavy Chains, Immunoglobulin Variable Region, Peptide Library, Pichia, Protein Engineering
Abstract

Yeast surface display is an efficient tool for isolating and engineering antibody fragments, both scFv and Fab. We describe the use of protein display on Pichia pastoris for the rapid selection of camelid antibodies composed only of heavy chains (nanobodies) from a library derived from a llama immunized with Green Fluorescent Protein. The library of nanobody-coding sequences was fused to the C-terminal part of the Saccharomyces cerevisiae alpha-agglutinin gene (SAG1) and expressed in glycoengineered P. pastoris. A high efficiency transformation protocol yielded a library of 5x10(7) clones. About 80% of the clones strongly expressed the nanobody fusion. Nanobody-displaying clones were rapidly enriched by fluorescence activated cell sorting (FACS), and GFP-specific nanobody-displaying clones were isolated and equilibrium dissociation constants (K(d)) determined. This technology for displaying protein libraries on P. pastoris enables the isolation and engineering of antibody-derived molecules in a robust eukaryotic expression host.

DOI10.1016/j.jbiotec.2009.10.010
Alternate JournalJ. Biotechnol.
PubMed ID19861136