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.Recent advances within the field of combinatorial protein engineering have led to the development of several complementary technologies for the selection of novel protein variants from large libraries. So far, phage display has been the preferred format for directed evolution efforts (39), but more recently, other techniques, such as ribosomal display (29), covalent display, and different formats of cell display, have become attractive alternatives (46). Cell surface display combined with fluorescence-activated cell sorting (FACS) constitutes a powerful strategy for isolation of novel ligands with improved affinity, stability, or enzymatic activity (4,28,37). The high throughput and quantitative multiparameter population analysis of modern flow cytometers makes FACS ideal for protein engineering applications (7,46). However, FACS sorting would only be applicable with cell display systems, since the phage particles are too small to be sorted with the present state-of-the-art flow cytometers (6, 43).Traditionally, engineering of antibody fragments has been the dominating strategy for generating novel proteins with specific ligand-binding properties (17). More recently, other protein scaffolds have also been investigated as sources for novel affinity ligands (26,38). One such novel class of affinity proteins, called affibody ligands, i.e., engineered Staphylococcus aureus protein A (SpA) domains, has recently been described (23). Combinatorial libraries were created through simultaneous randomization of 13 amino acid residues. Through the...

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

“…The extracted cell wall proteins were purified on HSA columns and analyzed using SDS–PAGE under reducing conditions (Fig. 2) [14]. The three mutant Z fusion proteins all migrated as full‐length proteins with a similar molecular weight as the previously characterized Z wt fusion protein (Fig.…”

“…It was demonstrated that cells expressing the ligand binding affibody could be enriched almost quantitatively from a moderate excess of non‐ligand binding cells by a single round of FACS. Extending the method to include two rounds of selection with reamplification by growth after the first round, target cells could be enriched 25,000‐fold from a model system consisting of one target cell in a million [14].…”

“…Since, in the previous study [14], S. carnosus cells displaying specific binding proteins were sorted from staphylococci displaying non‐binders, we here wanted to challenge the system to investigate whether fine‐affinity discrimination of closely related binders could be achieved, a feature that would be required for an efficient affinity‐based selection system. Four previously characterized IgG‐binding Z variants [32], exhibiting only small differences in target affinity, were expressed and analyzed in terms of surface accessibility and ligand binding in whole cell enzymatic assays.…”

Simplified characterization through site-specific protease-mediated release of affinity proteins selected by staphylococcal display

“…The surface display plasmid vector, pSCX:Z wt (Wernérus et al ., 2003), was used for construction of the new vectors. The vector pSCX:Z wt His 6 3C, hereafter denoted pHis3C, was created by ligating a sticky‐end fragment of annealed oligonucleotides, encoding the peptide H6EALFQGP, downstream of the Z wt domain in the vector pSCX:Z wt digested previously with SalI and HindIII restriction enzymes (Fermentas, Vilnius, Lithuania).…”

Affinity proteins and their generation

“…The successful applications of yeast and E. coli display in combinatorial protein engineering in the early 2000, and the potential of cell‐based systems to become attractive alternatives to phage display, inspired the evaluation of staphylococcal cell‐surface display also for this purpose. Employing a second‐generation display vector, the staphylococcal system was evaluated in a model library application using flow cytometry . The system was further optimized by the introduction of a surface expression normalization strategy using the ABP‐tag and fluorescently labeled albumin in order to improve the ability of discriminating between closely related binders …”