title: Fine affinity discrimination by normalized fluorescence activated cell sorting in staphylococcal surface display.
abstract: We have investigated a staphylococcal surface display system for its potential future use as a protein library display system in combinatorial biochemistry. Efficient affinity-based selections require a system capable of fine affinity discrimination of closely related binders to minimize the loss of potentially improved variants. In this study, a significant breakthrough was achieved to avoid biases due to potential cell-to-cell variations in surface expression levels, since it was found that a generic protein tag, present within the displayed recombinant surface proteins on the cells, could be successfully employed to obtain normalization of the target-binding signal. Four mutated variants of a staphylococcal protein A domain with different affinity to human IgG were successfully expressed on the surface of recombinant Staphylococcus carnosus cells. The system was evaluated for affinity-based cell sorting experiments, where cell-displayed protein A domains with an 8-fold difference in target affinity were mixed at a ratio of 1:1000 and sorted using FACS. Enrichment factors around 140-fold were obtained from a single round of sorting under normal library sorting conditions when the top 0.1% fraction having the highest antigen binding to surface expression level ratio was sorted. The results demonstrate that the system would have a potential as a selection system in protein library display applications, and the normalization strategy should indeed make it possible to achieve fine affinity discriminations in future library selections.
authors.affiliation: Department of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Center, Roslagstullsbacken 21, SE-106 91 Stockholm, Sweden.
authors.forename: John
authors.initials: J
authors.lastname: Löfblom
authors.forename: Henrik
authors.initials: H
authors.lastname: Wernérus
authors.forename: Stefan
authors.initials: S
authors.lastname: Ståhl
mesh_terms: D000595:Amino Acid Sequence; D000915:Antibody Affinity; D005434:Flow Cytometry / Q000379:methods*; D007074:Immunoglobulin G / Q000276:immunology; D008969:Molecular Sequence Data; D009154:Mutation; D019151:Peptide Library; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D016415:Sequence Alignment; D013205:Staphylococcal Protein A / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D013210:Staphylococcus / Q000378:metabolism*; D014169:Transformation, Bacterial
chemical_list: D007074:Immunoglobulin G; D019151:Peptide Library; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A