Fine affinity discrimination by normalized fluorescence activated cell sorting in staphylococcal surface display

• Published in: FEMS microbiology letters 2005-07, Vol.248 (2), p.189-198
• Main Authors: LÖFBLOM, John, WERNERUS, Henrik, STAHL, Stefan
• Format: Article
• Language: English
• Subjects: Affibody; Affinity; Affinity discrimination; Amino Acid Sequence; Antibody Affinity; Antigens; Bacteriology; Binders; Binding; Biologi; Biological and medical sciences; Biology; Cell and molecular biology; Cell surface display; Cell- och molekylärbiologi; Combinatorial analysis; Display devices; FACS; Flow cytometry; Flow Cytometry - methods; Fluorescence; Fundamental and applied biological sciences. Psychology; Genetics; Gram positive; Immunoglobulin G; Immunoglobulin G - immunology; Libraries; Microbiology; Molecular biology; Molecular Sequence Data; Molekylärbiologi; Mutation; NATURAL SCIENCES; NATURVETENSKAP; Peptide Library; Protein A; Protein folding; Protein Structure, Tertiary; Protein transport; Proteins; Recombinant Proteins - immunology; Recombinant Proteins - metabolism; Sequence Alignment; Staphylococcal Protein A - genetics; Staphylococcal Protein A - immunology; Staphylococcal Protein A - metabolism; Staphylococcus - metabolism; Staphylococcus carnosus; Transformation, Bacterial
• ISSN: 0378-1097; 1574-6968; 1574-6968
• DOI: 10.1016/j.femsle.2005.05.040

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.