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.

A truncated and dimeric format of an Affibody library on bacteria enables FACS‐mediated isolation of amyloid‐beta aggregation inhibitors with subnanomolar affinity

“…It has previously been demonstrated that staphylococcal cell surface display in combination with fluorescenceactivated cell sorting (FACS) is well suited for combinatorial affinity maturation efforts, since it allows for high precision affinity discrimination between binders [35][36][37][38]. Recently, the method was used for affinity maturation of a HER3-specific Affibody, and generated binders with picomolar affinities [39].…”

“…It has been shown that there is a correlation between the affinity of peripherally-administered Ab-specific agents and the efflux of Ab from the brain to the serum [29]. As levels of Ab peptides in the blood are low (subnanomolar) [30][31][32][33][34], it is probably critical to use capturing-agents of high affinity for such applications.It has previously been demonstrated that staphylococcal cell surface display in combination with fluorescenceactivated cell sorting (FACS) is well suited for combinatorial affinity maturation efforts, since it allows for high precision affinity discrimination between binders [35][36][37][38]. Recently, the method was used for affinity maturation of a HER3-specific Affibody, and generated binders with picomolar affinities [39].…”

Evaluation of Staphylococcal Cell Surface Display and Flow Cytometry for Postselectional Characterization of Affinity Proteins in Combinatorial Protein Engineering Applications

“…We have previously described a system for display of proteins and peptides on the cell surface of the gram-positive bacterium Staphylococcus carnosus (17, 36-39, 45, 51-55). The staphylococcal display system has recently been improved for protein engineering purposes (20), and optimization of the electroporation protocol has increased the transformation frequency to approximately 10 6 transformants per transformation (19), enabling the construction of large displayed combinatorial protein libraries. A 58-amino-acid, three-helical-bundle protein, derived from staphylococcal protein A (24), has been used as a protein engineering scaffold, and the randomized and selected affinity proteins are denoted affibody molecules (12,26,27,30).…”

“…In order to do this for the novel staphylococcal cell surface display method, we have determined the equilibrium dissociation constants (K D ) of three related affibody molecules (Z wt , Z N28A , and Z K35A ) (20) with affinity for human immunoglobulin G (IgG) by cell display together with flow cytometry and by biosensor analysis. Furthermore, the K D and the dissociation rate constant (k off ) of an albumin binding domain (ABD wt ) (44) for human serum albumin (HSA) have been determined using both methods.…”

Comparative characteristic of the methods of protein antigens epitope mapping

“…ДНК, що кодує досліджуваний протеїн-антиген, ампліфікується за допомогою полімеразної ланцюгової реакції та фрагментується за допомогою ультразву-ку до розмірів у 50-150 нуклеотидів, після чого одержані фрагменти ДНК клонуються у бактеріальний вектор (разом із послідовністю, що кодує альбумінзв'язувальний протеїн), яким клітини Staphylococcus carnosus транс-формуються шляхом електропорації. Таким чином, фрагменти антигену є «зшитими» із альбумінзв'язувальним протеїном, що відіграє роль молекулярного спейсера між клітинною стінкою та досліджуваним пептидом, а та-кож є важливим для подальшого сортування клітин у проточній цитофлуорометрії [46,47]. Синтезовані та експресовані на поверхні S. carnosus пептиди на наступному етапі взаємодіють із міченими антитілами та людсь-…”