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

• We have investigated a staphylococcal surface display system for its potential future use as a protein library display system ill 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. (c) 2005 Federation of European Microbiological Societies.

Ämnesord

NATURVETENSKAP  -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)

Nyckelord

Affibody; Affinity discrimination; Cell surface display; FACS; Gram positive; Staphylococcus carnosus; immunoglobulin G; membrane protein; antibody combining site; article; binding affinity; cell interaction; cellular distribution; evaluation; fluorescence activated cell sorting; nonhuman; priority journal; protein domain; protein expression; protein localization; protein targeting; signal transduction; Staphylococcus; Staphylococcus carnosus; surface property; Amino Acid Sequence; Antibody Affinity; Flow Cytometry; Immunoglobulin G; Molecular Sequence Data; Mutation; Peptide Library; Protein Structure

Tertiary; Recombinant Proteins; Sequence Alignment; Staphylococcal Protein A; Staphylococcus; Transformation

Bacterial; Posibacteria; Staphylococcus carnosus

Molecular biology

Molekylärbiologi

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