Abstract
We recently developed "autonomous hypermutation yeast surface display" (AHEAD), a technology that enables the rapid generation of potent and specific antibodies in yeast. AHEAD pairs yeast surface display with an error-prone orthogonal DNA replication system (OrthoRep) to continuously and rapidly mutate surface-displayed antibodies, thereby enabling enrichment for stronger binding variants through repeated rounds of cell growth and fluorescence-activated cell sorting. AHEAD currently utilizes a standard galactose induction system to drive the selective display of antibodies on the yeast surface. However, achieving maximal display levels can require up to 48 h of induction. Here we report an updated version of the AHEAD platform that utilizes a synthetic β-estradiol-induced gene expression system to regulate the surface display of antibodies and find that induction is notably faster in achieving surface display for both our AHEAD system and traditional yeast surface display from nuclear plasmids that do not hypermutate. The updated AHEAD platform was fully functional in repeated rounds of evolution to drive the rapid evolution of antibodies.
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