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<h2> Development of a novel strategy for engineering high-affinity proteins by yeast display </h2> <p> </p> <em><strong> S.A. Richman<sup>1</sup>, S.J. Healan<sup>1</sup>, K.S. Weber<sup>1</sup>, D.L. Donermeyer<sup>2</sup>, M.L. Dossett<sup>3</sup><sup>,4</sup>, P.D. Greenberg<sup>3</sup><sup>,4</sup>, P.M. Allen<sup>2</sup> and D.M. Kranz<sup>1</sup><sup>,5</sup></strong></em> <p> <em> <sup>1</sup> Department of Biochemistry, University of Illinois at Urbana-Champaign 600 S. Mathews Avenue, Urbana, IL 61801, USA <sup>2</sup> Department of Pathology and Immunology, Washington University School of Medicine St Louis, MO 63110, USA <sup>3</sup> Department of Immunology, University of Washington WA 98109, USA <sup>4</sup> The Division of Clinical Research, Fred Hutchinson Cancer Research Center 1100 Fairview Avenue North, Seattle, WA 98109, USA </em> </p> <p> <em>An open access article: Protein Engineering Design and Selection 2006 19(6):255-264; doi:10.1093/protein/gzl00. </em> </p> <p> </p> <p> <strong>Abstract</strong> </p> <p> Yeast display provides a system for engineering high-affinity<sup></sup>proteins using a fluorescent-labeled ligand and fluorescence-activated<sup></sup>cell sorting (FACS). In cases where it is difficult to obtain<sup></sup>purified ligands, or to access FACS instrumentation, an alternative<sup></sup>selection strategy would be useful. Here we show that yeast<sup></sup>expressing high-affinity proteins against a mammalian cell surface<sup></sup>ligand could be rapidly selected by density centrifugation.<sup></sup>Yeast cell–mammalian cell conjugates were retained at<sup></sup>the density interface, separated from unbound yeast. High-affinity<sup></sup>T cell receptors (TCRs) displayed on yeast were isolated using<sup></sup>antigen presenting cells that expressed TCR ligands, peptides<sup></sup>bound to products of the major histocompatibility complex (MHC).<sup></sup>The procedure yielded 1000-fold enrichments, in a single centrifugation,<sup></sup>of yeast displaying high-affinity TCRs. We defined the affinity<sup></sup>limits of the method and isolated high-affinity TCR mutants<sup></sup>against peptide variants that differed by only a single residue.<sup></sup>The approach was applied to TCRs specific for class I or class<sup></sup>II MHC, an important finding since peptide-class II MHC ligands<sup></sup>have been particularly difficult to purify. As yeast display<sup></sup>has also been used previously to identify antigen-specific antibodies,<sup></sup>the method should be applicable to the selection of antibodies,<sup></sup>as well as TCRs, with high-affinity for tumor cell-surface antigens.<sup></sup> </p> <p> </p> <p> <em>Keywords:</em> directed evolution/major histocompatibility complex/T cell receptor/yeast display </p> <p> </p>
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