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PEDS Advance Access originally published online on March 20, 2006
Protein Engineering Design and Selection 2006 19(6):255-264; doi:10.1093/protein/gzl008
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© The Author 2006. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org

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

S.A. Richman1, S.J. Healan1, K.S. Weber1, D.L. Donermeyer2, M.L. Dossett3,4, P.D. Greenberg3,4, P.M. Allen2 and D.M. Kranz1,5

1 Department of Biochemistry, University of Illinois at Urbana-Champaign 600 S. Mathews Avenue, Urbana, IL 61801, USA 2 Department of Pathology and Immunology, Washington University School of Medicine St Louis, MO 63110, USA 3 Department of Immunology, University of Washington WA 98109, USA 4 The Division of Clinical Research, Fred Hutchinson Cancer Research Center 1100 Fairview Avenue North, Seattle, WA 98109, USA

5Author to whom correspondence should be addressed. E-mail: d-kranz{at}uiuc.edu

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.

Keywords: directed evolution/major histocompatibility complex/T cell receptor/yeast display

Received October 30, 2005; revised February 8, 2006; accepted February 9, 2006.

Edited by Hennie Hoogenboom


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