Engineering Antibody Affinity by Yeast Surface Display

This chapter elaborates the engineering antibody affinity by yeast surface display (YSD). YSD is a powerful tool for engineering the affinity, specificity, and stability of antibodies, as well as other proteins. The methods for displaying an antibody on yeast, creating mutant libraries and sorting libraries for improved clones are presented. YSD involves the expression of a protein of interest on the yeast cell wall, where it can interact with proteins and small molecules in solution. The protein is expressed as a fusion to the Aga2p mating agglutinin protein, which is in turn linked by two disulfide bonds to the Aga1p protein linked covalently to the cell wall. Labeling yeast that are displaying an antibody or antibody library with a fluorescent or biotinylated antigen allows quantification of binding affinity, and enables library sorting by fluorescence-activated cell sorting. A second fluorophore conjugated to an antibody is used to detect the epitope tag C-terminal to the scFv, which allows for the normalization of expression and eliminates nondisplaying yeast from quantification. It is found that a complete cycle of mutagenesis and screening, from wild-type clone to improved mutant clone requires conservatively approximately 3–6 weeks.