374693 Enhancing Biomolecular Screening By Combining Yeast Surface Display with Noncanonical Amino Acids
Yeast surface display (YSD) is an established approach for identifying and improving the properties of proteins for use in diagnostic and therapeutic settings. In this work, we employ noncanonical amino acids (ncAAs) to enhance YSD by 1) enabling screening in a switchable display/secretion format and 2) introducing amino acids with reactive functional groups into displayed proteins. The use of switchable display/secretion enables yeast-based isolation of candidate binding proteins followed by immediate evaluation of the candidates’ biological properties in soluble form. The introduction of ncAAs with reactive side chains into displayed proteins provides facile approaches to the construction and evaluation of bioconjugates on the yeast surface.
To implement both of these applications, we developed a dual plasmid system enabling display of a protein containing a site-specifically incorporated ncAA. An antibody-like scFv-Fc protein containing an appropriately positioned amber codon was encoded in a display vector, while constitutively expressed E. coli amber suppression tRNAs and variants of the E. coli tyrosyl-tRNA synthetase were encoded in a suppression vector. This modular platform enabled confirmation of amber suppression events via flow cytometric detection of full-length scFv-Fcs displayed on the surface of yeast. To date, we have confirmed the incorporation of O-methyltyrosine, p-acetylphenylalanine, and p-azidophenylalanine into proteins displayed on the surface of yeast using appropriate aminoacyl-tRNA synthetase variants. Moreover, little to no full-length protein is displayed when ncAAs are omitted from the induction media, providing further support that ncAAs are being incorporated into the detected full-length proteins.
The ncAA-mediated display of scFv-Fcs on the yeast surface serves directly as a display/secretion system suitable for high-throughput biomolecular screening. Model enrichments using bead-based selections indicate that yeast displaying antibody-like scFv-Fcs specific for a target antigen can be enriched by approximately 500-fold in a single selection step, confirming the switchable system’s ability to efficiently identify binding proteins of interest. Initial secretion experiments have consistently resulted in the production of more than one milligram per liter of purified scFv-Fc, a sufficient quantity of protein for most basic biological assays. Encouraged by these results, we have constructed synthetic antibody libraries in the switchable format and used the libraries to identify scFv-Fcs recognizing epidermal growth factor receptor (EGFR), a validated cancer antigen. Product secreted from enriched populations specifically binds to cells overexpressing the membrane-bound form of EGFR, confirming both the success of our screens and recognition of the target antigen in its native form. These results suggest that future assays for additional biological functions (e.g., receptor clustering or downregulation, cellular proliferation, or cellular differentiation) are feasible with the system and should enable the rapid identification of proteins that modulate cellular behavior. The implementation of the switchable display/secretion technology enables cloning-free approaches to the generation of specific binding reagents and functional evaluation of these proteins for cancer targeting and other applications.
The development of a platform that controls YSD via a ncAA incorporation event also provided a starting point for the construction of bioconjugates on the yeast surface. After confirming the incorporation of ketone- and azide-containing ncAAs into proteins displayed on the surface of yeast, we began investigating the chemical modification of these functional groups. Flow cytometry experiments detecting reaction products have led to the identification of conditions enabling selective oxime ligations (ketones), copper-catalyzed azide-alkyne cycloadditions (azides), and strain-promoted cycloadditions (azides) on the surface of yeast. Thus, this approach enables both efficient display of proteins containing ncAAs and facile, site-specific protein modification, giving this system all of the necessary components for constructing bioconjugates on the surface of yeast. We anticipate that this approach will offer unprecedented throughput for the construction, evaluation, and directed evolution of antibody-drug conjugates and other small molecule-protein conjugates, thus enabling the development of therapeutics that simultaneously exploit the favorable properties of both proteins and small molecules.