Yeast cell surface display of proteins and uses thereof

摘要

The present invention provides a genetic method of tethering polypeptides to the yeast cell wall in a form accessible for binding to macromolecules. Combining this method with fluorescence-activated cell sorting provides a means of selecting proteins with increased or decreased affinity for another molecule, altered specificity, or conditional binding. As one embodiment, attaching an scFv antibody fragment to the Aga2p agglutinin effectively mimics the cell surface display of antibodies by B cells in the immune system for affinity maturation in vivo. As another embodiment, T cell receptor mutants can be isolated by this method that are efficiently displayed on the yeast cell surface, providing a means of altering T cell receptor binding affinity and specificity by library screening.

主权项

1. A method of producing a yeast cell displayed variant ligand binding protein with enhanced binding properties relative to a wild-type of said ligand binding protein, the method comprising:
isolating a gene encoding said wild-type binding protein; creating a library of mutated proteins by randomly mutating said wild-type protein; incorporating each said mutated protein into respective expression cassettes, each having the structure 5′-GAL 1-10 promoter—a Aga2p—mutated polypeptide; incorporating each said expression cassette into a respective vector; transforming yeast cells with said cassette-containing vectors to yield a multiplicity of transformed yeast cells; expressing said cassettes in said transformed yeast cells, whereby said ligand binding protein is displayed on the surface of each said yeast cell, said displayed ligand binding protein containing one of said mutated binding; labeling the displayed proteins on said yeast cells by binding a specific label to said displayed proteins; employing flow cytometry to sort said yeast cells according to their labeling characteristics; determining the surface expression level of said ligand binding protein in said sorted cells; determining the ligand binding characteristics of said ligand binding protein on the surface of said sorted cells whereby at least one preferred yeast cell expressing an abundance of ligand binding protein which exhibits enhanced ligand binding characteristics is identified; and cloning said at least one preferred yeast cell.