title: Fluorescence-activated cell sorting of specific affibody-displaying staphylococci.
abstract: Efficient enrichment of staphylococcal cells displaying specific heterologous affinity ligands on their cell surfaces was demonstrated by using fluorescence-activated cell sorting. Using bacterial surface display of peptide or protein libraries for the purpose of combinatorial protein engineering has previously been investigated by using gram-negative bacteria. Here, the potential for using a gram-positive bacterium was evaluated by employing the well-established surface expression system for Staphylococcus carnosus. Staphylococcus aureus protein A domains with binding specificity to immunoglobulin G or engineered specificity for the G protein of human respiratory syncytial virus were expressed as surface display on S. carnosus cells. The surface accessibility and retained binding specificity of expressed proteins were demonstrated in whole-cell enzyme and flow cytometry assays. Also, affibody-expressing target cells could be sorted essentially quantitatively from a moderate excess of background cells in a single step by using a high-stringency sorting mode. Furthermore, in a simulated library selection experiment, a more-than-25,000-fold enrichment of target cells could be achieved through only two rounds of cell sorting and regrowth. The results obtained indicate that staphylococcal surface display of affibody libraries combined with fluoresence-activated cell sorting might indeed constitute an attractive alternative to existing technology platforms for affinity-based selections.
authors.affiliation: Department of Biotechnology, AlbaNova University Center, Royal Institute of Technology (KTH), SE-106 91 Stockholm, Sweden.
authors.forename: Henrik
authors.initials: H
authors.lastname: Wernérus
authors.forename: Patrik
authors.initials: P
authors.lastname: Samuelson
authors.forename: Stefan
authors.initials: S
authors.lastname: Ståhl
mesh_terms: D000915:Antibody Affinity; D004926:Escherichia coli / Q000235:genetics; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D019151:Peptide Library; D010957:Plasmids; D015183:Restriction Mapping; D013205:Staphylococcal Protein A / Q000235:genetics*; D013210:Staphylococcus / Q000235:genetics / Q000276:immunology*; D013211:Staphylococcus aureus / Q000235:genetics / Q000276:immunology; D014170:Transformation, Genetic
chemical_list: D007074:Immunoglobulin G; D019151:Peptide Library; D013205:Staphylococcal Protein A