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Optimisation of recombinant TNFα production in Escherichia coli using GFP fusions and flow cytometry

Escherichia coli is commonly used industrially to manufacture recombinant proteins for biopharmaceutical applications, as well as in academic and industrial settings for R&D purposes. Optimisation of recombinant protein production remains problematic as many proteins are difficult to make, and p...

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Autores principales: Zulkifly, Nurul Asma Hasliza, Selas Castiñeiras, Tania, Overton, Tim W.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10433901/
https://www.ncbi.nlm.nih.gov/pubmed/37600304
http://dx.doi.org/10.3389/fbioe.2023.1171823
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author Zulkifly, Nurul Asma Hasliza
Selas Castiñeiras, Tania
Overton, Tim W.
author_facet Zulkifly, Nurul Asma Hasliza
Selas Castiñeiras, Tania
Overton, Tim W.
author_sort Zulkifly, Nurul Asma Hasliza
collection PubMed
description Escherichia coli is commonly used industrially to manufacture recombinant proteins for biopharmaceutical applications, as well as in academic and industrial settings for R&D purposes. Optimisation of recombinant protein production remains problematic as many proteins are difficult to make, and process conditions must be optimised for each individual protein. An approach to accelerate process development is the use of a green fluorescent protein (GFP) fusions, which can be used to rapidly and simply measure the quantity and folding state of the protein of interest. In this study, we used GFP fusions to optimise production of recombinant human protein tumour necrosis factor (rhTNFα) using a T7 expression system. Flow cytometry was used to measure fluorescence and cell viability on a single cell level to determine culture heterogeneity. Fluorescence measurements were found to be comparable to data generated by subcellular fractionation and SDS-PAGE, a far more time-intensive technique. We compared production of rhTNFα-GFP with that of GFP alone to determine the impact of rhTNFα on expression levels. Optimised shakeflask conditions were then transferred to fed-batch high cell density bioreactor cultures. Finally, the expression of GFP from a paraBAD expression vector was compared to the T7 system. We highlight the utility of GFP fusions and flow cytometry for rapid process development.
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spelling pubmed-104339012023-08-18 Optimisation of recombinant TNFα production in Escherichia coli using GFP fusions and flow cytometry Zulkifly, Nurul Asma Hasliza Selas Castiñeiras, Tania Overton, Tim W. Front Bioeng Biotechnol Bioengineering and Biotechnology Escherichia coli is commonly used industrially to manufacture recombinant proteins for biopharmaceutical applications, as well as in academic and industrial settings for R&D purposes. Optimisation of recombinant protein production remains problematic as many proteins are difficult to make, and process conditions must be optimised for each individual protein. An approach to accelerate process development is the use of a green fluorescent protein (GFP) fusions, which can be used to rapidly and simply measure the quantity and folding state of the protein of interest. In this study, we used GFP fusions to optimise production of recombinant human protein tumour necrosis factor (rhTNFα) using a T7 expression system. Flow cytometry was used to measure fluorescence and cell viability on a single cell level to determine culture heterogeneity. Fluorescence measurements were found to be comparable to data generated by subcellular fractionation and SDS-PAGE, a far more time-intensive technique. We compared production of rhTNFα-GFP with that of GFP alone to determine the impact of rhTNFα on expression levels. Optimised shakeflask conditions were then transferred to fed-batch high cell density bioreactor cultures. Finally, the expression of GFP from a paraBAD expression vector was compared to the T7 system. We highlight the utility of GFP fusions and flow cytometry for rapid process development. Frontiers Media S.A. 2023-08-02 /pmc/articles/PMC10433901/ /pubmed/37600304 http://dx.doi.org/10.3389/fbioe.2023.1171823 Text en Copyright © 2023 Zulkifly, Selas Castiñeiras and Overton. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Bioengineering and Biotechnology
Zulkifly, Nurul Asma Hasliza
Selas Castiñeiras, Tania
Overton, Tim W.
Optimisation of recombinant TNFα production in Escherichia coli using GFP fusions and flow cytometry
title Optimisation of recombinant TNFα production in Escherichia coli using GFP fusions and flow cytometry
title_full Optimisation of recombinant TNFα production in Escherichia coli using GFP fusions and flow cytometry
title_fullStr Optimisation of recombinant TNFα production in Escherichia coli using GFP fusions and flow cytometry
title_full_unstemmed Optimisation of recombinant TNFα production in Escherichia coli using GFP fusions and flow cytometry
title_short Optimisation of recombinant TNFα production in Escherichia coli using GFP fusions and flow cytometry
title_sort optimisation of recombinant tnfα production in escherichia coli using gfp fusions and flow cytometry
topic Bioengineering and Biotechnology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10433901/
https://www.ncbi.nlm.nih.gov/pubmed/37600304
http://dx.doi.org/10.3389/fbioe.2023.1171823
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