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

Protein Eng Des Sel. 2006 Jun;19(6):255-64. doi: 10.1093/protein/gzl008. Epub 2006 Mar 20.


Yeast display provides a system for engineering high-affinity proteins using a fluorescent-labeled ligand and fluorescence-activated cell sorting (FACS). In cases where it is difficult to obtain purified ligands, or to access FACS instrumentation, an alternative selection strategy would be useful. Here we show that yeast expressing high-affinity proteins against a mammalian cell surface ligand could be rapidly selected by density centrifugation. Yeast cell-mammalian cell conjugates were retained at the density interface, separated from unbound yeast. High-affinity T cell receptors (TCRs) displayed on yeast were isolated using antigen presenting cells that expressed TCR ligands, peptides bound to products of the major histocompatibility complex (MHC). The procedure yielded 1000-fold enrichments, in a single centrifugation, of yeast displaying high-affinity TCRs. We defined the affinity limits of the method and isolated high-affinity TCR mutants against peptide variants that differed by only a single residue. The approach was applied to TCRs specific for class I or class II MHC, an important finding since peptide-class II MHC ligands have been particularly difficult to purify. As yeast display has also been used previously to identify antigen-specific antibodies, the method should be applicable to the selection of antibodies, as well as TCRs, with high-affinity for tumor cell-surface antigens.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Differentiation / genetics
  • Cell Differentiation / physiology
  • Directed Molecular Evolution*
  • Flow Cytometry / methods
  • Fluorescent Dyes / chemistry
  • Fungal Proteins / chemistry
  • Fungal Proteins / genetics*
  • Fungal Proteins / metabolism
  • Gene Library
  • Histocompatibility Antigens Class II / chemistry
  • Histocompatibility Antigens Class II / genetics
  • Histocompatibility Antigens Class II / metabolism
  • Ligands
  • Major Histocompatibility Complex*
  • Mutation
  • Protein Engineering / methods*
  • Receptors, Antigen, T-Cell / genetics
  • Receptors, Antigen, T-Cell / isolation & purification
  • Receptors, Antigen, T-Cell / metabolism*
  • Yeasts / genetics*


  • Fluorescent Dyes
  • Fungal Proteins
  • Histocompatibility Antigens Class II
  • Ligands
  • Receptors, Antigen, T-Cell