Development of a novel strategy for engineering high-affinity proteins by yeast display

Area of Science:

• Biotechnology
• Protein Engineering
• Immunology

Background:

• Yeast display is a powerful tool for engineering high-affinity proteins using fluorescent ligands and fluorescence-activated cell sorting (FACS).
• Challenges arise when purified ligands are unavailable or FACS instrumentation is inaccessible, necessitating alternative selection strategies.

Purpose of the Study:

• To develop and validate an alternative selection method for high-affinity proteins using yeast display.
• To demonstrate the efficacy of density centrifugation for isolating yeast displaying high-affinity T cell receptors (TCRs) against mammalian cell surface ligands.

Main Methods:

• Yeast displaying high-affinity proteins were selected using density centrifugation by forming conjugates with mammalian cells expressing target ligands.
• High-affinity T cell receptors (TCRs) were isolated using antigen-presenting cells expressing TCR ligands, specifically peptides bound to major histocompatibility complex (MHC).

Main Results:

• Density centrifugation achieved rapid selection, yielding 1000-fold enrichment of yeast displaying high-affinity TCRs in a single step.
• The method successfully isolated high-affinity TCR mutants against single-residue peptide variants and was applied to TCRs specific for both class I and class II MHC.
• This approach proved effective even for difficult-to-purify peptide-class II MHC ligands.

Conclusions:

• Density centrifugation provides an efficient and accessible alternative to FACS for selecting high-affinity proteins via yeast display.
• The method is broadly applicable for isolating high-affinity T cell receptors (TCRs) and antibodies against various cell surface antigens, including challenging targets like peptide-MHC complexes.