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Yeast display provides a system for engineering high-affinity proteins using a fluorescent-labeled …


Biology Articles » Bioengineering » Development of a novel strategy for engineering high-affinity proteins by yeast display

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

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

An open access article: Protein Engineering Design and Selection 2006 19(6):255-264; doi:10.1093/protein/gzl00. 

 

Abstract

Yeast display provides a system for engineering high-affinityproteins using a fluorescent-labeled ligand and fluorescence-activatedcell sorting (FACS). In cases where it is difficult to obtainpurified ligands, or to access FACS instrumentation, an alternativeselection strategy would be useful. Here we show that yeastexpressing high-affinity proteins against a mammalian cell surfaceligand could be rapidly selected by density centrifugation.Yeast cell–mammalian cell conjugates were retained atthe density interface, separated from unbound yeast. High-affinityT cell receptors (TCRs) displayed on yeast were isolated usingantigen presenting cells that expressed TCR ligands, peptidesbound 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 affinitylimits of the method and isolated high-affinity TCR mutantsagainst peptide variants that differed by only a single residue.The approach was applied to TCRs specific for class I or classII MHC, an important finding since peptide-class II MHC ligandshave been particularly difficult to purify. As yeast displayhas 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

 


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