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The transient expression of recombinant proteins in plant cell packs facilitates stable isotope labelling for NMR spectroscopy
Nuclear magnetic resonance (NMR) spectroscopy can be used to determine the structure, dynamics and interactions of proteins. However, protein NMR requires stable isotope labelling for signal detection. The cells used for the production of recombinant proteins must therefore be grown in medium contai...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
John Wiley and Sons Inc.
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9491462/ https://www.ncbi.nlm.nih.gov/pubmed/35702941 http://dx.doi.org/10.1111/pbi.13873 |
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author | Opdensteinen, Patrick Sperl, Laura E. Mohamadi, Mariam Kündgen‐Redding, Nicole Hagn, Franz Buyel, Johannes Felix |
author_facet | Opdensteinen, Patrick Sperl, Laura E. Mohamadi, Mariam Kündgen‐Redding, Nicole Hagn, Franz Buyel, Johannes Felix |
author_sort | Opdensteinen, Patrick |
collection | PubMed |
description | Nuclear magnetic resonance (NMR) spectroscopy can be used to determine the structure, dynamics and interactions of proteins. However, protein NMR requires stable isotope labelling for signal detection. The cells used for the production of recombinant proteins must therefore be grown in medium containing isotopically labelled substrates. Stable isotope labelling is well established in Escherichia coli, but bacteria are only suitable for the production of simple proteins without post‐translational modifications. More complex proteins require eukaryotic production hosts, but their growth can be impaired by labelled media, thus reducing product yields and increasing costs. To address this limitation, we used media supplemented with isotope‐labelled substrates to cultivate the tobacco‐derived cell line BY‐2, which was then cast into plant cell packs (PCPs) for the transient expression of a labelled version of the model protein GB1. Mass spectrometry confirmed the feasibility of isotope labelling with (15)N and (2)H using this approach. The resulting NMR spectrum featured a signal dispersion comparable to recombinant GB1 produced in E. coli. PCPs therefore offer a rapid and cost‐efficient alternative for the production of isotope‐labelled proteins for NMR analysis, especially suitable for complex proteins that cannot be produced in microbial systems. |
format | Online Article Text |
id | pubmed-9491462 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | John Wiley and Sons Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-94914622022-09-30 The transient expression of recombinant proteins in plant cell packs facilitates stable isotope labelling for NMR spectroscopy Opdensteinen, Patrick Sperl, Laura E. Mohamadi, Mariam Kündgen‐Redding, Nicole Hagn, Franz Buyel, Johannes Felix Plant Biotechnol J Research Articles Nuclear magnetic resonance (NMR) spectroscopy can be used to determine the structure, dynamics and interactions of proteins. However, protein NMR requires stable isotope labelling for signal detection. The cells used for the production of recombinant proteins must therefore be grown in medium containing isotopically labelled substrates. Stable isotope labelling is well established in Escherichia coli, but bacteria are only suitable for the production of simple proteins without post‐translational modifications. More complex proteins require eukaryotic production hosts, but their growth can be impaired by labelled media, thus reducing product yields and increasing costs. To address this limitation, we used media supplemented with isotope‐labelled substrates to cultivate the tobacco‐derived cell line BY‐2, which was then cast into plant cell packs (PCPs) for the transient expression of a labelled version of the model protein GB1. Mass spectrometry confirmed the feasibility of isotope labelling with (15)N and (2)H using this approach. The resulting NMR spectrum featured a signal dispersion comparable to recombinant GB1 produced in E. coli. PCPs therefore offer a rapid and cost‐efficient alternative for the production of isotope‐labelled proteins for NMR analysis, especially suitable for complex proteins that cannot be produced in microbial systems. John Wiley and Sons Inc. 2022-07-19 2022-10 /pmc/articles/PMC9491462/ /pubmed/35702941 http://dx.doi.org/10.1111/pbi.13873 Text en © 2022 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd. https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ (https://creativecommons.org/licenses/by-nc-nd/4.0/) License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made. |
spellingShingle | Research Articles Opdensteinen, Patrick Sperl, Laura E. Mohamadi, Mariam Kündgen‐Redding, Nicole Hagn, Franz Buyel, Johannes Felix The transient expression of recombinant proteins in plant cell packs facilitates stable isotope labelling for NMR spectroscopy |
title | The transient expression of recombinant proteins in plant cell packs facilitates stable isotope labelling for NMR spectroscopy |
title_full | The transient expression of recombinant proteins in plant cell packs facilitates stable isotope labelling for NMR spectroscopy |
title_fullStr | The transient expression of recombinant proteins in plant cell packs facilitates stable isotope labelling for NMR spectroscopy |
title_full_unstemmed | The transient expression of recombinant proteins in plant cell packs facilitates stable isotope labelling for NMR spectroscopy |
title_short | The transient expression of recombinant proteins in plant cell packs facilitates stable isotope labelling for NMR spectroscopy |
title_sort | transient expression of recombinant proteins in plant cell packs facilitates stable isotope labelling for nmr spectroscopy |
topic | Research Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9491462/ https://www.ncbi.nlm.nih.gov/pubmed/35702941 http://dx.doi.org/10.1111/pbi.13873 |
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