The use of scFv-displaying yeast in mammalian cell surface selections.
Yeast surface display has proven to be a powerful tool for the directed evolution of immunological proteins when soluble ligands are available (Cho, B.K., Kieke, M.C., Boder, E.T., Wittrup, K.D., Kranz, D.M., 1998. A yeast surface display system for the discovery of ligands that trigger cell activation. J. Immunol. Methods 220, 179; Boder, E.T., Midelfort, K.S., Wittrup, K.D., 2000. Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity. Proc. Natl. Acad. Sci. U. S. A. 97, 10701; Shusta, E.V., Holler, P.D., Kieke, M.C., Kranz, D.M., Wittrup, K.D., 2000. Directed evolution of a stable scaffold for T-cell receptor engineering. Nat. Biotechnol. 18, 754; Esteban, O., Zhao, H., 2004. Directed evolution of soluble single-chain human class II MHC molecules. J. Mol. Biol. 340, 81). This investigation extends the utility of this display platform by demonstrating its capacity for use in cell panning selections. This was accomplished by employing a model single-chain antibody (scFv)-hapten system that allowed for detailed investigation of the factors governing panning success. Yeast displaying anti-fluorescein scFv (4-4-20) exhibited specific interactions with the fluoresceinated endothelial cells and could be recovered from large backgrounds of irrelevant yeast in just three rounds. Successful selections required as few as 1700 fluorescein ligands per cell, and a three-round enrichment ratio of 10(6) was possible. These results indicate that yeast surface display is a viable option for use in cell or tissue-based selections.
Wang
Xin Xiang
XX
Department of Chemical and Biological Engineering, University of Wisconsin-Madison, 1415 Engineering Drive, Madison, WI 53706, USA.
Shusta
Eric V
EV
D000818:Animals; D000915:Antibody Affinity; D002450:Cell Communication / Q000276:immunology; D002460:Cell Line; D002469:Cell Separation / Q000379:methods; D016650:Fluorescein-5-isothiocyanate; D006801:Humans; D007128:Immunoglobulin Fragments / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000378:metabolism*; D008024:Ligands; D019151:Peptide Library; D051381:Rats; D011990:Receptors, Transferrin / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000276:immunology* / Q000378:metabolism
D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D008024:Ligands; D019151:Peptide Library; D011990:Receptors, Transferrin; D016650:Fluorescein-5-isothiocyanate