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The iSplit GFP assay detects intracellular recombinant proteins in Bacillus subtilis
BACKGROUND: Bacillus subtilis is one of the most important microorganisms for recombinant protein production. It possesses the GRAS (generally recognized as safe) status and a potent protein secretion capacity. Secretory protein production greatly facilitates downstream processing and thus significa...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8419962/ https://www.ncbi.nlm.nih.gov/pubmed/34488765 http://dx.doi.org/10.1186/s12934-021-01663-7 |
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author | Lenz, Patrick Hilgers, Fabienne Burmeister, Alina Zimmermann, Leonie Volkenborn, Kristina Grünberger, Alexander Kohlheyer, Dietrich Drepper, Thomas Jaeger, Karl-Erich Knapp, Andreas |
author_facet | Lenz, Patrick Hilgers, Fabienne Burmeister, Alina Zimmermann, Leonie Volkenborn, Kristina Grünberger, Alexander Kohlheyer, Dietrich Drepper, Thomas Jaeger, Karl-Erich Knapp, Andreas |
author_sort | Lenz, Patrick |
collection | PubMed |
description | BACKGROUND: Bacillus subtilis is one of the most important microorganisms for recombinant protein production. It possesses the GRAS (generally recognized as safe) status and a potent protein secretion capacity. Secretory protein production greatly facilitates downstream processing and thus significantly reduces costs. However, not all heterologous proteins are secreted and intracellular production poses difficulties for quantification. To tackle this problem, we have established a so-called intracellular split GFP (iSplit GFP) assay in B. subtilis as a tool for the in vivo protein detection during expression in batch cultures and at a single-cell level. For the iSplit GFP assay, the eleventh β-sheet of sfGFP is fused to a target protein and can complement a detector protein consisting of the respective truncated sfGFP (GFP1-10) to form fluorescent holo-GFP. RESULTS: As proof of concept, the GFP11-tag was fused C-terminally to the E. coli β-glucuronidase GUS, resulting in fusion protein GUS11. Variable GUS and GUS11 production levels in B. subtilis were achieved by varying the ribosome binding site via spacers of increasing lengths (4–12 nucleotides) for the GUS-encoding gene. Differences in intracellular enzyme accumulation were determined by measuring the GUS11 enzymatic activity and subsequently by adding the detector protein to respective cell extracts. Moreover, the detector protein was co-produced with the GUS11 using a two-plasmid system, which enabled the in vivo detection and online monitoring of glucuronidase production. Using this system in combination with flow cytometry and microfluidics, we were able to monitor protein production at a single-cell level thus yielding information about intracellular protein distribution and culture heterogeneity. CONCLUSION: Our results demonstrate that the iSplit GFP assay is suitable for the detection, quantification and online monitoring of recombinant protein production in B. subtilis during cultivation as well as for analyzing production heterogeneity and intracellular localization at a single-cell level. GRAPHIC ABSTRACT: [Image: see text] SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12934-021-01663-7. |
format | Online Article Text |
id | pubmed-8419962 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-84199622021-09-09 The iSplit GFP assay detects intracellular recombinant proteins in Bacillus subtilis Lenz, Patrick Hilgers, Fabienne Burmeister, Alina Zimmermann, Leonie Volkenborn, Kristina Grünberger, Alexander Kohlheyer, Dietrich Drepper, Thomas Jaeger, Karl-Erich Knapp, Andreas Microb Cell Fact Research BACKGROUND: Bacillus subtilis is one of the most important microorganisms for recombinant protein production. It possesses the GRAS (generally recognized as safe) status and a potent protein secretion capacity. Secretory protein production greatly facilitates downstream processing and thus significantly reduces costs. However, not all heterologous proteins are secreted and intracellular production poses difficulties for quantification. To tackle this problem, we have established a so-called intracellular split GFP (iSplit GFP) assay in B. subtilis as a tool for the in vivo protein detection during expression in batch cultures and at a single-cell level. For the iSplit GFP assay, the eleventh β-sheet of sfGFP is fused to a target protein and can complement a detector protein consisting of the respective truncated sfGFP (GFP1-10) to form fluorescent holo-GFP. RESULTS: As proof of concept, the GFP11-tag was fused C-terminally to the E. coli β-glucuronidase GUS, resulting in fusion protein GUS11. Variable GUS and GUS11 production levels in B. subtilis were achieved by varying the ribosome binding site via spacers of increasing lengths (4–12 nucleotides) for the GUS-encoding gene. Differences in intracellular enzyme accumulation were determined by measuring the GUS11 enzymatic activity and subsequently by adding the detector protein to respective cell extracts. Moreover, the detector protein was co-produced with the GUS11 using a two-plasmid system, which enabled the in vivo detection and online monitoring of glucuronidase production. Using this system in combination with flow cytometry and microfluidics, we were able to monitor protein production at a single-cell level thus yielding information about intracellular protein distribution and culture heterogeneity. CONCLUSION: Our results demonstrate that the iSplit GFP assay is suitable for the detection, quantification and online monitoring of recombinant protein production in B. subtilis during cultivation as well as for analyzing production heterogeneity and intracellular localization at a single-cell level. GRAPHIC ABSTRACT: [Image: see text] SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12934-021-01663-7. BioMed Central 2021-09-06 /pmc/articles/PMC8419962/ /pubmed/34488765 http://dx.doi.org/10.1186/s12934-021-01663-7 Text en © The Author(s) 2021 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 Lenz, Patrick Hilgers, Fabienne Burmeister, Alina Zimmermann, Leonie Volkenborn, Kristina Grünberger, Alexander Kohlheyer, Dietrich Drepper, Thomas Jaeger, Karl-Erich Knapp, Andreas The iSplit GFP assay detects intracellular recombinant proteins in Bacillus subtilis |
title | The iSplit GFP assay detects intracellular recombinant proteins in Bacillus subtilis |
title_full | The iSplit GFP assay detects intracellular recombinant proteins in Bacillus subtilis |
title_fullStr | The iSplit GFP assay detects intracellular recombinant proteins in Bacillus subtilis |
title_full_unstemmed | The iSplit GFP assay detects intracellular recombinant proteins in Bacillus subtilis |
title_short | The iSplit GFP assay detects intracellular recombinant proteins in Bacillus subtilis |
title_sort | isplit gfp assay detects intracellular recombinant proteins in bacillus subtilis |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8419962/ https://www.ncbi.nlm.nih.gov/pubmed/34488765 http://dx.doi.org/10.1186/s12934-021-01663-7 |
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