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Engineering Saccharomyces cerevisiae for the production and secretion of Affibody molecules
BACKGROUND: Affibody molecules are synthetic peptides with a variety of therapeutic and diagnostic applications. To date, Affibody molecules have mainly been produced by the bacterial production host Escherichia coli. There is an interest in exploring alternative production hosts to identify potenti...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8905840/ https://www.ncbi.nlm.nih.gov/pubmed/35264156 http://dx.doi.org/10.1186/s12934-022-01761-0 |
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author | Gast, Veronica Sandegren, Anna Dunås, Finn Ekblad, Siri Güler, Rezan Thorén, Staffan Tous Mohedano, Marta Molin, Mikael Engqvist, Martin K. M. Siewers, Verena |
author_facet | Gast, Veronica Sandegren, Anna Dunås, Finn Ekblad, Siri Güler, Rezan Thorén, Staffan Tous Mohedano, Marta Molin, Mikael Engqvist, Martin K. M. Siewers, Verena |
author_sort | Gast, Veronica |
collection | PubMed |
description | BACKGROUND: Affibody molecules are synthetic peptides with a variety of therapeutic and diagnostic applications. To date, Affibody molecules have mainly been produced by the bacterial production host Escherichia coli. There is an interest in exploring alternative production hosts to identify potential improvements in terms of yield, ease of production and purification advantages. In this study, we evaluated the feasibility of Saccharomyces cerevisiae as a production chassis for this group of proteins. RESULTS: We examined the production of three different Affibody molecules in S. cerevisiae and found that these Affibody molecules were partially degraded. An albumin-binding domain, which may be attached to the Affibody molecules to increase their half-life, was identified to be a substrate for several S. cerevisiae proteases. We tested the removal of three vacuolar proteases, proteinase A, proteinase B and carboxypeptidase Y. Removal of one of these, proteinase A, resulted in intact secretion of one of the targeted Affibody molecules. Removal of either or both of the two additional proteases, carboxypeptidase Y and proteinase B, resulted in intact secretion of the two remaining Affibody molecules. The produced Affibody molecules were verified to bind their target, human HER3, as potently as the corresponding molecules produced in E. coli in an in vitro surface-plasmon resonance binding assay. Finally, we performed a fed-batch fermentation with one of the engineered protease-deficient S. cerevisiae strains and achieved a protein titer of 530 mg Affibody molecule/L. CONCLUSION: This study shows that engineered S. cerevisiae has a great potential as a production host for recombinant Affibody molecules, reaching a high titer, and for proteins where endotoxin removal could be challenging, the use of S. cerevisiae obviates the need for endotoxin removal from protein produced in E. coli. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12934-022-01761-0. |
format | Online Article Text |
id | pubmed-8905840 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-89058402022-03-18 Engineering Saccharomyces cerevisiae for the production and secretion of Affibody molecules Gast, Veronica Sandegren, Anna Dunås, Finn Ekblad, Siri Güler, Rezan Thorén, Staffan Tous Mohedano, Marta Molin, Mikael Engqvist, Martin K. M. Siewers, Verena Microb Cell Fact Research BACKGROUND: Affibody molecules are synthetic peptides with a variety of therapeutic and diagnostic applications. To date, Affibody molecules have mainly been produced by the bacterial production host Escherichia coli. There is an interest in exploring alternative production hosts to identify potential improvements in terms of yield, ease of production and purification advantages. In this study, we evaluated the feasibility of Saccharomyces cerevisiae as a production chassis for this group of proteins. RESULTS: We examined the production of three different Affibody molecules in S. cerevisiae and found that these Affibody molecules were partially degraded. An albumin-binding domain, which may be attached to the Affibody molecules to increase their half-life, was identified to be a substrate for several S. cerevisiae proteases. We tested the removal of three vacuolar proteases, proteinase A, proteinase B and carboxypeptidase Y. Removal of one of these, proteinase A, resulted in intact secretion of one of the targeted Affibody molecules. Removal of either or both of the two additional proteases, carboxypeptidase Y and proteinase B, resulted in intact secretion of the two remaining Affibody molecules. The produced Affibody molecules were verified to bind their target, human HER3, as potently as the corresponding molecules produced in E. coli in an in vitro surface-plasmon resonance binding assay. Finally, we performed a fed-batch fermentation with one of the engineered protease-deficient S. cerevisiae strains and achieved a protein titer of 530 mg Affibody molecule/L. CONCLUSION: This study shows that engineered S. cerevisiae has a great potential as a production host for recombinant Affibody molecules, reaching a high titer, and for proteins where endotoxin removal could be challenging, the use of S. cerevisiae obviates the need for endotoxin removal from protein produced in E. coli. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12934-022-01761-0. BioMed Central 2022-03-09 /pmc/articles/PMC8905840/ /pubmed/35264156 http://dx.doi.org/10.1186/s12934-022-01761-0 Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data. |
spellingShingle | Research Gast, Veronica Sandegren, Anna Dunås, Finn Ekblad, Siri Güler, Rezan Thorén, Staffan Tous Mohedano, Marta Molin, Mikael Engqvist, Martin K. M. Siewers, Verena Engineering Saccharomyces cerevisiae for the production and secretion of Affibody molecules |
title | Engineering Saccharomyces cerevisiae for the production and secretion of Affibody molecules |
title_full | Engineering Saccharomyces cerevisiae for the production and secretion of Affibody molecules |
title_fullStr | Engineering Saccharomyces cerevisiae for the production and secretion of Affibody molecules |
title_full_unstemmed | Engineering Saccharomyces cerevisiae for the production and secretion of Affibody molecules |
title_short | Engineering Saccharomyces cerevisiae for the production and secretion of Affibody molecules |
title_sort | engineering saccharomyces cerevisiae for the production and secretion of affibody molecules |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8905840/ https://www.ncbi.nlm.nih.gov/pubmed/35264156 http://dx.doi.org/10.1186/s12934-022-01761-0 |
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