Cellular and Molecular Life Sciences

, Volume 70, Issue 6, pp 1081–1093 | Cite as

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

  • Filippa Fleetwood
  • Nick Devoogdt
  • Mireille Pellis
  • Ulrich Wernery
  • Serge Muyldermans
  • Stefan Ståhl
  • John Löfblom
Research article

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 107 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.

Keywords

Bacterial display Combinatorial protein engineering Nanobodies Phage display Recombinant antibodies 

Supplementary material

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Copyright information

© Springer Basel 2012

Authors and Affiliations

  • Filippa Fleetwood
    • 1
  • Nick Devoogdt
    • 2
    • 3
  • Mireille Pellis
    • 2
    • 4
  • Ulrich Wernery
    • 5
  • Serge Muyldermans
    • 2
    • 4
  • Stefan Ståhl
    • 1
  • John Löfblom
    • 1
  1. 1.Division of Molecular Biotechnology, School of Biotechnology, KTH, Royal Institute of TechnologyAlbaNova University CenterStockholmSweden
  2. 2.Laboratory of Cellular and Molecular ImmunologyVrije Universiteit BrusselBrusselsBelgium
  3. 3.Laboratory of In Vivo Cellular and Molecular ImagingVrije Universiteit BrusselBrusselsBelgium
  4. 4.Department of Structural BiologyVIBBrusselsBelgium
  5. 5.Central Veterinary Research Laboratory (CVRL)DubaiUnited Arab Emirates

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