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A Protein-Engineered, Enhanced Yeast Display Platform for Rapid Evolution of Challenging Targets

Abstract : 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°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.
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Contributor : Laurence Langlais Connect in order to contact the contributor
Submitted on : Wednesday, January 12, 2022 - 9:40:00 AM
Last modification on : Friday, August 5, 2022 - 12:03:10 PM
Long-term archiving on: : Wednesday, April 13, 2022 - 6:27:36 PM


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Distributed under a Creative Commons Attribution 4.0 International License




Jiří Zahradník, Debabrata Dey, Shir Marciano, Lucie Kolářová, Chloé I Charendoff, et al.. A Protein-Engineered, Enhanced Yeast Display Platform for Rapid Evolution of Challenging Targets. ACS Synthetic Biology, 2021, 10 (12), pp.3445-3460. ⟨10.1021/acssynbio.1c00395⟩. ⟨pasteur-03522398⟩



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