Rapidly Inducible Yeast Surface Display for Antibody Evolution with OrthoRep.

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 ß-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.

Rapidly inducible yeast surface display for antibody evolution with OrthoRep.

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 (FACS). 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 hours of induction. Here, we report an updated version of the AHEAD platform that utilizes a synthetic ß-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 as well as 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.

Protein Engineering and High-Throughput Screening by Yeast Surface Display: Survey of Current Methods.

Yeast surface display (YSD) is a powerful tool in biotechnology that links genotype to phenotype. In this review, the latest advancements in protein engineering and high-throughput screening based on YSD are covered. The focus is on innovative methods for overcoming challenges in YSD in the context of biotherapeutic drug discovery and diagnostics. Topics ranging from titrating avidity in YSD using transcriptional control to the development of serological diagnostic assays relying on serum biopanning and mitigation of unspecific binding are covered. Screening techniques against nontraditional cellular antigens, such as cell lysates, membrane proteins, and extracellular matrices are summarized and techniques are further delved into for expansion of the chemical repertoire, considering protein-small molecule hybrids and noncanonical amino acid incorporation. Additionally, in vivo gene diversification and continuous evolution in yeast is discussed. Collectively, these techniques enhance the diversity and functionality of engineered proteins isolated via YSD, broadening the scope of applications that can be addressed. The review concludes with future perspectives and potential impact of these advancements on protein engineering. The goal is to provide a focused summary of recent progress in the field.