Optimized Yeast Surface Display Workflow for Directed Evolution of Proteases

Directed evolution mimics the natural evolutionary process in a fast, controlled laboratory environment to evolve proteins with desirable functions or traits. Screening a large pool of mutant protein-coding genes allows functional selection of desired variants for applications in biotechnology, medicine, and synthetic biology. Using this protocol, we perform directed evolution in Saccharomyces cerevisiae to engineer protease variants with high specificity for a non-native substrate sequence. We display these protease variants on the yeast cell surface to facilitate the selection of desired variants via fluorescence-activated cell sorting (FACS) over multiple rounds of enrichment. Four key elements of the substrate cassette are co-expressed with the protease library: a fusion protein composed of (i) the yeast adhesion receptor subunit Aga2, (ii) selection and (iii) counter-selection substrate sequences, (iv) epitope tag sequences, and an optional endoplasmic reticulum (ER) retrieval signal sequence. Yeast cells in which only the selection substrate sequence is cleaved are isolated using multicolor FACS via fluorescent (phycoerythrin/fluorescein isothiocyanate-tagged) anti-epitope antibodies. Here we provide details of the optimized, step-by-step protocol to conduct a protease evolution campaign, including DNA insert library design and substrate cassette design, yeast cell electroporation with high transformation efficiency (up to 10⁹ transformants per microgram of DNA), FACS-based selection, enrichment, and sequencing of evolved variants. We demonstrate our directed evolution protocol by evolving the tobacco etch virus nuclear inclusion A protease (TEVp) from cleaving its natural substrate sequence, ENLYFQ↓S, to cleaving a new sequence, ENLYFE↓S.