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Optimized Expression and Isolation of Recombinant Active Secreted Proteases Using Pichia pastoris
Recombinant proteins of high quality are crucial starting materials for all downstream applications, but the inherent complexities of proteins and their expression and purification create significant challenges. The Pichia pastoris yeast is a highly useful eukaryotic protein expression system. Pichi...
Autores principales: | , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Bio-Protocol
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9993078/ https://www.ncbi.nlm.nih.gov/pubmed/36908634 http://dx.doi.org/10.21769/BioProtoc.4628 |
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author | Lanser, Dylan M. Gelli, Angie |
author_facet | Lanser, Dylan M. Gelli, Angie |
author_sort | Lanser, Dylan M. |
collection | PubMed |
description | Recombinant proteins of high quality are crucial starting materials for all downstream applications, but the inherent complexities of proteins and their expression and purification create significant challenges. The Pichia pastoris yeast is a highly useful eukaryotic protein expression system. Pichia’s low cost, genetic tractability, rapid gene expression, and scalability make it an ideal expression system for foreign proteins. Here, we developed a protocol that has optimized the expression and isolation of a non-mammalian secreted metalloprotease, where we can routinely generate recombinant proteins that are pure and proteolytically active. We maximized growth and protein production by altering the feeding regime, through implementation of a non-fermentable and non-repressing carbon source during the methanol-induction phase. This approach increased biomass production and yielded milligrams of recombinant protein. Downstream applications involving active, recombinant fungal proteases, such as conjugation to nanoparticles and structure-related studies, are greatly facilitated with this improved, standardized approach. Graphical abstract [Image: see text] |
format | Online Article Text |
id | pubmed-9993078 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Bio-Protocol |
record_format | MEDLINE/PubMed |
spelling | pubmed-99930782023-03-09 Optimized Expression and Isolation of Recombinant Active Secreted Proteases Using Pichia pastoris Lanser, Dylan M. Gelli, Angie Bio Protoc Methods Article Recombinant proteins of high quality are crucial starting materials for all downstream applications, but the inherent complexities of proteins and their expression and purification create significant challenges. The Pichia pastoris yeast is a highly useful eukaryotic protein expression system. Pichia’s low cost, genetic tractability, rapid gene expression, and scalability make it an ideal expression system for foreign proteins. Here, we developed a protocol that has optimized the expression and isolation of a non-mammalian secreted metalloprotease, where we can routinely generate recombinant proteins that are pure and proteolytically active. We maximized growth and protein production by altering the feeding regime, through implementation of a non-fermentable and non-repressing carbon source during the methanol-induction phase. This approach increased biomass production and yielded milligrams of recombinant protein. Downstream applications involving active, recombinant fungal proteases, such as conjugation to nanoparticles and structure-related studies, are greatly facilitated with this improved, standardized approach. Graphical abstract [Image: see text] Bio-Protocol 2023-03-05 /pmc/articles/PMC9993078/ /pubmed/36908634 http://dx.doi.org/10.21769/BioProtoc.4628 Text en Copyright © 2023 The Authors; exclusive licensee Bio-protocol LLC. https://creativecommons.org/licenses/by-nc/4.0/This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/). |
spellingShingle | Methods Article Lanser, Dylan M. Gelli, Angie Optimized Expression and Isolation of Recombinant Active Secreted Proteases Using Pichia pastoris |
title | Optimized Expression and Isolation of Recombinant Active Secreted Proteases Using Pichia pastoris |
title_full | Optimized Expression and Isolation of Recombinant Active Secreted Proteases Using Pichia pastoris |
title_fullStr | Optimized Expression and Isolation of Recombinant Active Secreted Proteases Using Pichia pastoris |
title_full_unstemmed | Optimized Expression and Isolation of Recombinant Active Secreted Proteases Using Pichia pastoris |
title_short | Optimized Expression and Isolation of Recombinant Active Secreted Proteases Using Pichia pastoris |
title_sort | optimized expression and isolation of recombinant active secreted proteases using pichia pastoris |
topic | Methods Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9993078/ https://www.ncbi.nlm.nih.gov/pubmed/36908634 http://dx.doi.org/10.21769/BioProtoc.4628 |
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