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

S. A. Richman, S. J. Healan, K. S. Weber, D. L. Donermeyer, M. L. Dossett, P. D. Greenberg, P. M. Allen, David M Kranz

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)255-264
Number of pages10
JournalProtein Engineering, Design and Selection
Volume19
Issue number6
DOIs
StatePublished - Jun 1 2006

Fingerprint

Fungal Proteins
T-cells
Yeast
T-Cell Antigen Receptor
Yeasts
Display devices
Proteins
Ligands
Cells
Major Histocompatibility Complex
Peptides
Centrifugation
Antigens
Sorting
Antibodies
Flow Cytometry
Fluorescence
Antigen-Presenting Cells
Surface Antigens
Tumors

Keywords

  • Directed evolution
  • Major histocompatibility complex
  • T cell receptor
  • Yeast display

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Molecular Biology

Cite this

Richman, S. A., Healan, S. J., Weber, K. S., Donermeyer, D. L., Dossett, M. L., Greenberg, P. D., ... Kranz, D. M. (2006). Development of a novel strategy for engineering high-affinity proteins by yeast display. Protein Engineering, Design and Selection, 19(6), 255-264. https://doi.org/10.1093/protein/gzl008

Development of a novel strategy for engineering high-affinity proteins by yeast display. / Richman, S. A.; Healan, S. J.; Weber, K. S.; Donermeyer, D. L.; Dossett, M. L.; Greenberg, P. D.; Allen, P. M.; Kranz, David M.

In: Protein Engineering, Design and Selection, Vol. 19, No. 6, 01.06.2006, p. 255-264.

Research output: Contribution to journalArticle

Richman, SA, Healan, SJ, Weber, KS, Donermeyer, DL, Dossett, ML, Greenberg, PD, Allen, PM & Kranz, DM 2006, 'Development of a novel strategy for engineering high-affinity proteins by yeast display', Protein Engineering, Design and Selection, vol. 19, no. 6, pp. 255-264. https://doi.org/10.1093/protein/gzl008
Richman, S. A. ; Healan, S. J. ; Weber, K. S. ; Donermeyer, D. L. ; Dossett, M. L. ; Greenberg, P. D. ; Allen, P. M. ; Kranz, David M. / Development of a novel strategy for engineering high-affinity proteins by yeast display. In: Protein Engineering, Design and Selection. 2006 ; Vol. 19, No. 6. pp. 255-264.
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