PEDS Advance Access published online on January 31, 2008
Protein Engineering Design and Selection, doi:10.1093/protein/gzm090
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A novel affinity protein selection system based on staphylococcal cell surface display and flow cytometry
1Department of Molecular Biotechnology, School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Center, SE-106 91 Stockholm 2 Affibody AB, Box 20137, SE-161 02 Bromma, Sweden
3 To whom correspondence should be addressed. E-mail: stefans{at}biotech.kth.se
Here we describe the first reported use of a Gram-positive bacterial system for the selection of affinity proteins from large combinatorial libraries displayed on the surface of Staphylococcus carnosus. An affibody library of 3 x 109 variants, based on a 58 residue domain from staphylococcal protein A, was pre-enriched for binding to human tumor necrosis factor-alpha (TNF-alpha) using one cycle of phage display and thereafter transferred to the staphylococcal host (
106 variants). The staphylococcal-displayed library was subjected to three rounds of flow-cytometric sorting, and the selected clones were screened and ranked by on-cell analysis for binding to TNF-alpha and further characterized using biosensor analysis and circular dichroism spectroscopy. The successful sorting yielded three different high-affinity binders (ranging from 95 pM to 2.2 nM) and constitutes the first selection of a novel affinity protein using Gram-positive bacterial display. The method combines the simplicity of working with a bacterial host with the advantages of displaying recombinant proteins on robust Gram-positive bacteria as well as using powerful flow cytometry in the selection and characterization process.
Keywords: affibody/cell surface display/combinatorial protein engineering/Gram-positive bacteria/Staphylococcus carnosus
Received December 12, 2007; revised December 12, 2007; accepted December 17, 2007.
These authors contributed equally to this work.