Engineering antibody affinity by yeast surface display.

Yeast surface display (YSD) is a powerful tool for engineering the affinity, specificity, and stability of antibodies, as well as other proteins. Since first described in 1997 by Boder and Wittrup, YSD has been employed successfully in engineering a number of antibodies, as well as T-cell receptors. A recently reported large nonimmune single chain antibody library serves as a good starting point for engineering high-affinity antibodies. Cloned variable genes from hybridomas or scFvs or Fabs from phage display libraries are also incorporated easily into a yeast display format. The original YSD protocols were described earlier, but new and refined methods have been developed, in particular improved vectors, mutagenesis methods, and efficient ligation-free yeast transformation procedures. This article provides up-to-date protocols for engineering single chain antibodies by YSD. Compared to other display formats, yeast surface display offers several advantages. One chief advantage to engineering protein affinity by YSD is that yeast cells can be sorted by fluorescence-activated cell sorting (FACS), allowing quantitative discrimination between mutants. Further, FACS simultaneously gives analysis data, eliminating the need for separate steps of expression and analysis after each round of sorting. Without exception to date, equilbrium-binding constants and dissociation rate constants measured for yeast-displayed proteins are in quantitative agreement with those measured for the same proteins in vitro using BIAcore or ELISA.