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A Protein-Engineered, Enhanced Yeast Display Platform for Rapid Evolution of Challenging Targets
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SYSNO ASEP 0556045 Document Type J - Journal Article R&D Document Type Journal Article Subsidiary J Článek ve WOS Title A Protein-Engineered, Enhanced Yeast Display Platform for Rapid Evolution of Challenging Targets Author(s) Zahradnik, J. (IL)
Dey, D. (IL)
Marciano, S. (IL)
Kolářová, Lucie (BTO-N)
Charendoff, C. (FR)
Subtil, A. (FR)
Schreiber, G. (IL)Number of authors 7 Source Title ACS Synthetic Biology. - : American Chemical Society - ISSN 2161-5063
Roč. 10, č. 12 (2021), s. 3445-3460Number of pages 16 s. Language eng - English Country US - United States Keywords green fluorescent protein ; surface display ; saccharomyces-cerevisiae ; directed evolution ; rf cloning Subject RIV EB - Genetics ; Molecular Biology OECD category Biochemical research methods Method of publishing Open access Institutional support BTO-N - RVO:86652036 UT WOS 000753341200018 EID SCOPUS 85120439372 DOI 10.1021/acssynbio.1c00395 Annotation Here, we enhanced the popular yeast display method by multiple rounds of DNA and protein engineering. We introduced surface exposure-tailored reporters, eUnaG2 and DnbALFA, creating a new platform of C and N terminal fusion vectors. The optimization of eUnaG2 resulted in five times brighter fluorescence and 10 degrees C increased thermostability than UnaG. The optimized DnbALFA has 10-fold the level of expression of the starting protein. Following this, different plasmids were developed to create a complex platform allowing a broad range of protein expression organizations and labeling strategies. Our platform showed up to five times better separation between nonexpressing and expressing cells compared with traditional pCTcon2 and c-myc labeling, allowing for fewer rounds of selection and achieving higher binding affinities. Testing 16 different proteins, the enhanced system showed consistently stronger expression signals over c-myc labeling. In addition to gains in simplicity, speed, and cost-effectiveness, new applications were introduced to monitor protein surface exposure and protein retention in the secretion pathway that enabled successful protein engineering of hard-to-express proteins. As an example, we show how we optimized the WD40 domain of the ATG16L1 protein for yeast surface and soluble bacterial expression, starting from a nonexpressing protein. As a second example, we show how using the here-presented enhanced yeast display method we rapidly selected high-affinity binders toward two protein targets, demonstrating the simplicity of generating new protein-protein interactions. While the methodological changes are incremental, it results in a qualitative enhancement in the applicability of yeast display for many applications. Workplace Institute of Biotechnology Contact Monika Kopřivová, Monika.Koprivova@ibt.cas.cz, Tel.: 325 873 700 Year of Publishing 2023 Electronic address https://pubs.acs.org/doi/10.1021/acssynbio.1c00395
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