A novel affinity protein selection system based on staphylococcal cell surface display and flow cytometry

• Published in: Protein engineering, design and selection design and selection, 2008-04, Vol.21 (4), p.247-255
• Main Authors: Kronqvist, Nina, Löfblom, John, Jonsson, Andreas, Wernérus, Henrik, Ståhl, Stefan
• Format: Article
• Language: English
• Subjects: affibody; affibody/cell surface display/combinatorial protein engineering/Gram-positive bacteria/Staphylococcus carnosus; Amino Acid Sequence; Bioengineering; Biosensing Techniques; Bioteknik; cell surface display; Circular Dichroism; combinatorial protein engineering; Flow Cytometry - methods; Gram-positive bacteria; Humans; Molecular Sequence Data; Peptide Library; Protein Binding; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - isolation & purification; Recombinant Fusion Proteins - metabolism; Staphylococcal Protein A - genetics; Staphylococcal Protein A - metabolism; Staphylococcus - genetics; Staphylococcus - metabolism; Staphylococcus carnosus; TECHNOLOGY; TEKNIKVETENSKAP; Tumor Necrosis Factor-alpha - chemistry; Tumor Necrosis Factor-alpha - metabolism
• ISSN: 1741-0126; 1741-0134; 1741-0134
• DOI: 10.1093/protein/gzm090

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 × 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.