Surface display of a single-domain antibody library on Gram-positive bacteria.

TitleSurface display of a single-domain antibody library on Gram-positive bacteria.
Publication TypeJournal Article
Year of Publication2013
AuthorsFleetwood, F., N. Devoogdt, M. Pellis, U. Wernery, S. Muyldermans, S. Ståhl, and J. Löfblom
JournalCell Mol Life Sci
Volume70
Issue6
Pagination1081-93
Date Published2013 Mar
ISSN1420-9071
KeywordsAntibody Affinity, Cell Surface Display Techniques, Cloning, Molecular, Directed Molecular Evolution, Flow Cytometry, Green Fluorescent Proteins, Peptide Library, Protein Engineering, Single-Domain Antibodies, Staphylococcus
Abstract

Combinatorial protein engineering for selection of proteins with novel functions, such as enzymes and affinity reagents, is an important tool in biotechnology, drug discovery, and other biochemical fields. Bacterial display is an emerging technology for isolation of new affinity proteins from such combinatorial libraries. Cells have certain properties that are attractive for directed evolution purposes, in particular the option to use quantitative flow-cytometric cell sorting for selection of binders. Here, an immune library of around 10(7) camelid single-domain antibody fragments (Nanobodies) was displayed on both the Gram-positive bacterium Staphylococcus carnosus and on phage. As demonstrated for the first time, the antibody repertoire was found to be well expressed on the bacterial surface and flow-cytometric sorting yielded a number of Nanobodies with subnanomolar affinity for the target protein, green fluorescent protein (GFP). Interestingly, the staphylococcal output repertoire and the binders from the phage display selection contained two slightly different sets of clones, containing both unique as well as several similar variants. All of the Nanobodies from the staphylococcal selection were also shown to enhance the fluorescence of GFP upon binding, potentially due to the fluorescence-based sorting principle. Our study highlights the impact of the chosen display technology on the variety of selected binders and thus the value of having alternative methods available, and demonstrates in addition that the staphylococcal system is suitable for generation of high-affinity antibody fragments.

DOI10.1007/s00018-012-1179-y
Alternate JournalCell. Mol. Life Sci.
PubMed ID23064703