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A Protein-Engineered, Enhanced Yeast Display Platform for Rapid Evolution of Challenging Targets
[Image: see text] 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...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Chemical Society
2021
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8689690/ https://www.ncbi.nlm.nih.gov/pubmed/34809429 http://dx.doi.org/10.1021/acssynbio.1c00395 |
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author | Zahradník, Jiří Dey, Debabrata Marciano, Shir Kolářová, Lucie Charendoff, Chloé I. Subtil, Agathe Schreiber, Gideon |
author_facet | Zahradník, Jiří Dey, Debabrata Marciano, Shir Kolářová, Lucie Charendoff, Chloé I. Subtil, Agathe Schreiber, Gideon |
author_sort | Zahradník, Jiří |
collection | PubMed |
description | [Image: see text] 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. |
format | Online Article Text |
id | pubmed-8689690 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | American Chemical Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-86896902021-12-22 A Protein-Engineered, Enhanced Yeast Display Platform for Rapid Evolution of Challenging Targets Zahradník, Jiří Dey, Debabrata Marciano, Shir Kolářová, Lucie Charendoff, Chloé I. Subtil, Agathe Schreiber, Gideon ACS Synth Biol [Image: see text] 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. American Chemical Society 2021-11-22 2021-12-17 /pmc/articles/PMC8689690/ /pubmed/34809429 http://dx.doi.org/10.1021/acssynbio.1c00395 Text en © 2021 The Authors. Published by American Chemical Society https://creativecommons.org/licenses/by/4.0/Permits the broadest form of re-use including for commercial purposes, provided that author attribution and integrity are maintained (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Zahradník, Jiří Dey, Debabrata Marciano, Shir Kolářová, Lucie Charendoff, Chloé I. Subtil, Agathe Schreiber, Gideon A Protein-Engineered, Enhanced Yeast Display Platform for Rapid Evolution of Challenging Targets |
title | A Protein-Engineered, Enhanced Yeast Display Platform
for Rapid Evolution of Challenging Targets |
title_full | A Protein-Engineered, Enhanced Yeast Display Platform
for Rapid Evolution of Challenging Targets |
title_fullStr | A Protein-Engineered, Enhanced Yeast Display Platform
for Rapid Evolution of Challenging Targets |
title_full_unstemmed | A Protein-Engineered, Enhanced Yeast Display Platform
for Rapid Evolution of Challenging Targets |
title_short | A Protein-Engineered, Enhanced Yeast Display Platform
for Rapid Evolution of Challenging Targets |
title_sort | protein-engineered, enhanced yeast display platform
for rapid evolution of challenging targets |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8689690/ https://www.ncbi.nlm.nih.gov/pubmed/34809429 http://dx.doi.org/10.1021/acssynbio.1c00395 |
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