Titratable Avidity Reduction Enhances Affinity Discrimination in Mammalian Cellular Selections of Yeast-Displayed Ligands.
Yeast surface display selections against mammalian cell monolayers have proven effective in isolating proteins with novel binding activity. Recent advances in this technique allow for the recovery of clones with even micromolar binding affinities. However, no efficient method has been shown for affinity-based selection in this context. This study demonstrates the effectiveness of titratable avidity reduction using dithiothreitol to achieve this goal. A series of epidermal growth factor receptor binding fibronectin domains with a range of affinities are used to quantitatively identify the number of ligands per yeast cell that yield the strongest selectivity between strong, moderate, and weak affinities. Notably, reduction of ligand display to 3,000-6,000 ligands per yeast cell of a 2 nM binder yields 16-fold better selectivity than that to a 17 nM binder. These lessons are applied to affinity maturation of an EpCAM-binding fibronectin population, yielding an enriched pool of ligands with significantly stronger affinity than that of an analogous pool sorted by standard cellular selection methods. Collectively, this study offers a facile approach for affinity selection of yeast-displayed ligands against full-length cellular targets and demonstrates the effectiveness of this method by generating EpCAM-binding ligands that are promising for further applications.
Department of Chemical Engineering and Materials Science and ‡Department of Medicinal Chemistry, University of Minnesota-Twin Cities , Minneapolis, Minnesota 55455, United States.
Lawrence A
LA
Stern
Department of Chemical Engineering and Materials Science and ‡Department of Medicinal Chemistry, University of Minnesota-Twin Cities , Minneapolis, Minnesota 55455, United States.
Clifford M
CM
Csizmar
Department of Chemical Engineering and Materials Science and ‡Department of Medicinal Chemistry, University of Minnesota-Twin Cities , Minneapolis, Minnesota 55455, United States.
Daniel R
DR
Woldring
Department of Chemical Engineering and Materials Science and ‡Department of Medicinal Chemistry, University of Minnesota-Twin Cities , Minneapolis, Minnesota 55455, United States.
Carston R
0000-0001-7927-719X
CR
Wagner
Department of Chemical Engineering and Materials Science and ‡Department of Medicinal Chemistry, University of Minnesota-Twin Cities , Minneapolis, Minnesota 55455, United States.
Benjamin J
0000-0003-3561-9463
BJ
Hackel
D000818:Animals; D045744:Cell Line, Tumor; D004229:Dithiothreitol; D000071858:Epithelial Cell Adhesion Molecule / Q000235:genetics / Q000378:metabolism; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D005353:Fibronectins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007202:Indicators and Reagents; D008024:Ligands; D051379:Mice; D010084:Oxidation-Reduction; D019151:Peptide Library; D011485:Protein Binding; D000072417:Protein Domains; D015202:Protein Engineering; D017186:Titrimetry; D015003:Yeasts / Q000235:genetics
D000071858:Epithelial Cell Adhesion Molecule; D005353:Fibronectins; D007202:Indicators and Reagents; D008024:Ligands; D019151:Peptide Library; D066246:ErbB Receptors; D004229:Dithiothreitol
combinatorial library screening; fibronectin domain; ligand discovery; protein engineering