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Lactiplantibacillus plantarum as a novel platform for production and purification of integral membrane proteins using RseP as the benchmark

The present study describes a detailed procedure for expressing and purifying the integral membrane protein RseP using the pSIP system and Lactiplantibacillus plantarum as an expression host. RseP is a membrane-bound site-2-protease and a known antibacterial target in multiple human pathogens. In th...

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Autores principales: Kristensen, Sofie S., Lukassen, Marie V., Siebenhaar, Suzana, Diep, Dzung B., Morth, J. Preben, Mathiesen, Geir
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10474122/
https://www.ncbi.nlm.nih.gov/pubmed/37658186
http://dx.doi.org/10.1038/s41598-023-41559-7
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author Kristensen, Sofie S.
Lukassen, Marie V.
Siebenhaar, Suzana
Diep, Dzung B.
Morth, J. Preben
Mathiesen, Geir
author_facet Kristensen, Sofie S.
Lukassen, Marie V.
Siebenhaar, Suzana
Diep, Dzung B.
Morth, J. Preben
Mathiesen, Geir
author_sort Kristensen, Sofie S.
collection PubMed
description The present study describes a detailed procedure for expressing and purifying the integral membrane protein RseP using the pSIP system and Lactiplantibacillus plantarum as an expression host. RseP is a membrane-bound site-2-protease and a known antibacterial target in multiple human pathogens. In the present study, we screened five RseP orthologs from Gram-positive bacteria and found RseP from Enterococcus faecium (EfmRseP) to yield the highest protein levels. The production conditions were optimized and EfmRseP was purified by immobilized metal ion affinity chromatography followed by size-exclusion chromatography. The purification resulted in an overall yield of approximately 1 mg of pure protein per 3 g of wet-weight cell pellet. The structural integrity of the purified protein was confirmed using circular dichroism. We further assessed the expression and purification of RseP from E. faecium in the Gram-negative Escherichia coli. Detection of soluble protein failed in two of the three E. coli strains tested. Purification of EfmRseP expressed in E. coli C43(DE3) resulted in a protein with lower purity compared to EfmRseP expressed in L. plantarum. To our knowledge, this is the first time L. plantarum and the pSIP expression system have been applied for the production of membrane proteins.
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spelling pubmed-104741222023-09-03 Lactiplantibacillus plantarum as a novel platform for production and purification of integral membrane proteins using RseP as the benchmark Kristensen, Sofie S. Lukassen, Marie V. Siebenhaar, Suzana Diep, Dzung B. Morth, J. Preben Mathiesen, Geir Sci Rep Article The present study describes a detailed procedure for expressing and purifying the integral membrane protein RseP using the pSIP system and Lactiplantibacillus plantarum as an expression host. RseP is a membrane-bound site-2-protease and a known antibacterial target in multiple human pathogens. In the present study, we screened five RseP orthologs from Gram-positive bacteria and found RseP from Enterococcus faecium (EfmRseP) to yield the highest protein levels. The production conditions were optimized and EfmRseP was purified by immobilized metal ion affinity chromatography followed by size-exclusion chromatography. The purification resulted in an overall yield of approximately 1 mg of pure protein per 3 g of wet-weight cell pellet. The structural integrity of the purified protein was confirmed using circular dichroism. We further assessed the expression and purification of RseP from E. faecium in the Gram-negative Escherichia coli. Detection of soluble protein failed in two of the three E. coli strains tested. Purification of EfmRseP expressed in E. coli C43(DE3) resulted in a protein with lower purity compared to EfmRseP expressed in L. plantarum. To our knowledge, this is the first time L. plantarum and the pSIP expression system have been applied for the production of membrane proteins. Nature Publishing Group UK 2023-09-01 /pmc/articles/PMC10474122/ /pubmed/37658186 http://dx.doi.org/10.1038/s41598-023-41559-7 Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open Access This 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/) .
spellingShingle Article
Kristensen, Sofie S.
Lukassen, Marie V.
Siebenhaar, Suzana
Diep, Dzung B.
Morth, J. Preben
Mathiesen, Geir
Lactiplantibacillus plantarum as a novel platform for production and purification of integral membrane proteins using RseP as the benchmark
title Lactiplantibacillus plantarum as a novel platform for production and purification of integral membrane proteins using RseP as the benchmark
title_full Lactiplantibacillus plantarum as a novel platform for production and purification of integral membrane proteins using RseP as the benchmark
title_fullStr Lactiplantibacillus plantarum as a novel platform for production and purification of integral membrane proteins using RseP as the benchmark
title_full_unstemmed Lactiplantibacillus plantarum as a novel platform for production and purification of integral membrane proteins using RseP as the benchmark
title_short Lactiplantibacillus plantarum as a novel platform for production and purification of integral membrane proteins using RseP as the benchmark
title_sort lactiplantibacillus plantarum as a novel platform for production and purification of integral membrane proteins using rsep as the benchmark
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10474122/
https://www.ncbi.nlm.nih.gov/pubmed/37658186
http://dx.doi.org/10.1038/s41598-023-41559-7
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