期刊
ACS COMBINATORIAL SCIENCE
卷 19, 期 5, 页码 315-323出版社
AMER CHEMICAL SOC
DOI: 10.1021/acscombsci.6b00191
关键词
ligand discovery; protein engineering; fibronectin domain; combinatorial library screening
资金
- National Institutes of Health [R21 EB019518, R21 CA185627, F30 CA210345]
- University of Minnesota
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 betwen 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.
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