A novel affinity protein selection system based on staphylococcal cell surface display and flow cytometry
2008 (English)In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 21, no 4, 247-255 p.Article in journal (Refereed) Published
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.
Place, publisher, year, edition, pages
2008. Vol. 21, no 4, 247-255 p.
affibody/cell surface display/combinatorial protein engineering/Gram-positive bacteria/Staphylococcus carnosus
IdentifiersURN: urn:nbn:se:kth:diva-7846DOI: 10.1093/protein/gzm090ISI: 000254295200004OAI: oai:DiVA.org:kth-7846DiVA: diva2:12987
QC 201007222007-12-142007-12-142010-12-06Bibliographically approved