Fluorescence-activated cell sorting of specific affibody-displaying staphylococci

Area of Science:

• Microbiology
• Biotechnology
• Protein Engineering

Background:

• Bacterial surface display is a powerful tool for protein engineering, primarily explored in gram-negative bacteria.
• Gram-positive bacteria, like Staphylococcus, offer alternative systems for surface display applications.

Purpose of the Study:

• To evaluate the potential of Staphylococcus carnosus, a gram-positive bacterium, for surface display of affinity ligands.
• To demonstrate efficient enrichment of cells displaying specific heterologous affinity ligands using fluorescence-activated cell sorting (FACS).

Main Methods:

• Utilized the established surface expression system in Staphylococcus carnosus.
• Expressed Staphylococcus aureus protein A domains with known (IgG) and engineered (RSV G protein) specificities on the bacterial surface.
• Employed whole-cell enzyme assays and flow cytometry to confirm surface accessibility and binding specificity.
• Applied high-stringency FACS for quantitative cell sorting and enrichment.

Main Results:

• Successfully displayed functional affinity ligands (Staphylococcus protein A domains) on the surface of Staphylococcus carnosus.
• Demonstrated retained binding specificity of the displayed proteins to their targets (IgG and RSV G protein).
• Achieved highly efficient enrichment of target cells (over 25,000-fold in two rounds) using FACS, even from excess background cells.

Conclusions:

• Staphylococcal surface display combined with FACS is a viable and attractive alternative for affinity-based protein library selection.
• This method offers efficient enrichment, potentially surpassing existing technology platforms for protein engineering and discovery.