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Construction and Application of a Plasmid-Based Signal Peptide Library for Improved Secretion of Recombinant Proteins with Priestia megaterium
The secretion of recombinant proteins plays an important role in their economic production and purification. The secretion efficiency depends on the responsible signal peptide (SP) in combination with the target protein and the given host and cannot be predicted so far. Due to its high plasmid stabi...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9032162/ https://www.ncbi.nlm.nih.gov/pubmed/35456829 http://dx.doi.org/10.3390/microorganisms10040777 |
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author | Mayer, Janine Knuuti, Tobias Baumgarten, Lisa Menke, Elise Bischoff, Lena Bunk, Boyke Biedendieck, Rebekka |
author_facet | Mayer, Janine Knuuti, Tobias Baumgarten, Lisa Menke, Elise Bischoff, Lena Bunk, Boyke Biedendieck, Rebekka |
author_sort | Mayer, Janine |
collection | PubMed |
description | The secretion of recombinant proteins plays an important role in their economic production and purification. The secretion efficiency depends on the responsible signal peptide (SP) in combination with the target protein and the given host and cannot be predicted so far. Due to its high plasmid stability, the lack of alkaline extracellular proteases and only few contaminating extracellular host proteins, Priestia megaterium provides a promising alternative to common Bacillus species. For the development of an easy and fast cloning and screening system to identify the SP best suited to a distinct protein, a plasmid-based SP library containing all predicted 182 Sec-dependent SPs from P. megaterium was established. The splitting of the SPs into 10 groups of individual multi-SP plasmids (pMSPs) allows their grouped amplification and application in screening approaches. The functionality of the whole library was demonstrated by enhancing the amount of the already well-secreted α-amylase AmyE by 1.6-fold. The secretion of a novel penicillin G acylase, which remained as insoluble protein inside the cells, as its native SP is unsuitable for secretion in P. megaterium, could be enhanced even up to 29-fold. Overall, only around 170 recombinant P. megaterium clones based on 50 inserted SPs had to be screened to achieve sufficient amounts for further enzyme characterizations. Thus, this newly developed plasmid-based genetic tool applicable for P. megaterium and also other Bacillus species facilitates the identification of suitable SPs for secretion of recombinant proteins. |
format | Online Article Text |
id | pubmed-9032162 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-90321622022-04-23 Construction and Application of a Plasmid-Based Signal Peptide Library for Improved Secretion of Recombinant Proteins with Priestia megaterium Mayer, Janine Knuuti, Tobias Baumgarten, Lisa Menke, Elise Bischoff, Lena Bunk, Boyke Biedendieck, Rebekka Microorganisms Article The secretion of recombinant proteins plays an important role in their economic production and purification. The secretion efficiency depends on the responsible signal peptide (SP) in combination with the target protein and the given host and cannot be predicted so far. Due to its high plasmid stability, the lack of alkaline extracellular proteases and only few contaminating extracellular host proteins, Priestia megaterium provides a promising alternative to common Bacillus species. For the development of an easy and fast cloning and screening system to identify the SP best suited to a distinct protein, a plasmid-based SP library containing all predicted 182 Sec-dependent SPs from P. megaterium was established. The splitting of the SPs into 10 groups of individual multi-SP plasmids (pMSPs) allows their grouped amplification and application in screening approaches. The functionality of the whole library was demonstrated by enhancing the amount of the already well-secreted α-amylase AmyE by 1.6-fold. The secretion of a novel penicillin G acylase, which remained as insoluble protein inside the cells, as its native SP is unsuitable for secretion in P. megaterium, could be enhanced even up to 29-fold. Overall, only around 170 recombinant P. megaterium clones based on 50 inserted SPs had to be screened to achieve sufficient amounts for further enzyme characterizations. Thus, this newly developed plasmid-based genetic tool applicable for P. megaterium and also other Bacillus species facilitates the identification of suitable SPs for secretion of recombinant proteins. MDPI 2022-04-05 /pmc/articles/PMC9032162/ /pubmed/35456829 http://dx.doi.org/10.3390/microorganisms10040777 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Mayer, Janine Knuuti, Tobias Baumgarten, Lisa Menke, Elise Bischoff, Lena Bunk, Boyke Biedendieck, Rebekka Construction and Application of a Plasmid-Based Signal Peptide Library for Improved Secretion of Recombinant Proteins with Priestia megaterium |
title | Construction and Application of a Plasmid-Based Signal Peptide Library for Improved Secretion of Recombinant Proteins with Priestia megaterium |
title_full | Construction and Application of a Plasmid-Based Signal Peptide Library for Improved Secretion of Recombinant Proteins with Priestia megaterium |
title_fullStr | Construction and Application of a Plasmid-Based Signal Peptide Library for Improved Secretion of Recombinant Proteins with Priestia megaterium |
title_full_unstemmed | Construction and Application of a Plasmid-Based Signal Peptide Library for Improved Secretion of Recombinant Proteins with Priestia megaterium |
title_short | Construction and Application of a Plasmid-Based Signal Peptide Library for Improved Secretion of Recombinant Proteins with Priestia megaterium |
title_sort | construction and application of a plasmid-based signal peptide library for improved secretion of recombinant proteins with priestia megaterium |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9032162/ https://www.ncbi.nlm.nih.gov/pubmed/35456829 http://dx.doi.org/10.3390/microorganisms10040777 |
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