Isolation of anti-T cell receptor scFv mutants by yeast surface display

Yeast surface display and sorting by flow cytometry have been used to isolate mutants of an scFv that is specific for the V beta 8 region of the T cell receptor, Selection was based on equilibrium binding by two fluorescently labeled probes, a soluble vps domain and an antibody to the c-myc epitope tag present at the carboxy-terminus of the scFv. The mutants that were selected in this screen included a scFv with threefold increased affinity for the V beta 8 and scFv clones that were bound with reduced affinities by the antic-myc antibody, The latter finding indicates that the yeast display system may be used to map conformational epitopes, which cannot be revealed by standard peptide screens. Equilibrium antigen binding constants were estimated within the surface display format, allowing screening of isolated mutants without necessitating subcloning and soluble expression. Only a relatively small library of yeast cells (3x10(5)) displaying randomly mutagenized scFv was screened to identify these mutants, indicating that this system will provide a powerful tool for engineering the binding properties of eucaryotic secreted and cell surface proteins.