Display of a naïve affibody library on staphylococci for selection of binders by means of flow cytometry sorting

• DOI: 10.1016/j.bbrc.2023.03.003
• Publication Date: 2023-03-04T01:15:41Z
• Authors: Charles Dahlsson ...; Anna Mestre Borras; Andreas Jonsson; Magdalena Malm; Nina Kronqvist; Filippa Fleetwood; Lisa Sandersjöö; Mathias Uhlén; John Löfblom; Stefan Ståhl; Hanna Lindberg
• Keywords: Peptide Library; Flow Cytometry; Protein Engineering; Protein Binding

Within the field of combinatorial protein engineering there is a great demand for robust high-throughput selection platforms that allow unbiased library display, affinity-based screening, and amplification selected clones. We have previously described development staphylococcal display system used displaying both alternative-scaffolds antibody-derived proteins. In this study, objective was to generate an improved expression vector screening high-complexity naïve affibody library, facilitate downstream validation isolated A high-affinity normalization tag, consisting two ABD-moieties, introduced simplify off-rate procedures. addition, furnished with TEV protease substrate recognition sequence upstream which enables proteolytic processing displayed construct binding signal. design, 13 58 surface-exposed amino acid positions were full randomization (except proline cysteine) using trinucleotide technology. The genetic successfully transformed Staphylococcus carnosus cells, generating exceeding 109 members. De novo selections against three target proteins (CD14, MAPK9 ZEGFR:2377) performed magnetic bead-based capture followed by flow-cytometric sorting, yielding molecules their respective nanomolar affinity. Taken together, results demonstrate feasibility proposed procedure new high