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Screening of Membrane Protein Production by Comparison of Transient Expression in Insect and Mammalian Cells

Membrane proteins are difficult biomolecules to express and purify. In this paper, we compare the small-scale production of six selected eukaryotic integral membrane proteins in insect and mammalian cell expression systems using different techniques for gene delivery. The target proteins were C term...

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Autores principales: Kaipa, Jagan Mohan, Krasnoselska, Ganna, Owens, Raymond J., van den Heuvel, Joop
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10216445/
https://www.ncbi.nlm.nih.gov/pubmed/37238687
http://dx.doi.org/10.3390/biom13050817
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author Kaipa, Jagan Mohan
Krasnoselska, Ganna
Owens, Raymond J.
van den Heuvel, Joop
author_facet Kaipa, Jagan Mohan
Krasnoselska, Ganna
Owens, Raymond J.
van den Heuvel, Joop
author_sort Kaipa, Jagan Mohan
collection PubMed
description Membrane proteins are difficult biomolecules to express and purify. In this paper, we compare the small-scale production of six selected eukaryotic integral membrane proteins in insect and mammalian cell expression systems using different techniques for gene delivery. The target proteins were C terminally fused to the green fluorescent marker protein GFP to enable sensitive monitoring. We show that the choice of expression systems makes a considerable difference to the yield and quality of the six selected membrane proteins. Virus-free transient gene expression (TGE) in insect High Five cells combined with solubilization in dodecylmaltoside plus cholesteryl hemisuccinate generated the most homogeneous samples for all six targets. Further, the affinity purification of the solubilized proteins using the Twin-Strep(®) tag improved protein quality in terms of yield and homogeneity compared to His-tag purification. TGE in High Five insect cells offers a fast and economically attractive alternative to the established methods that require either baculovirus construction and the infection of the insect cells or relatively expensive transient gene expression in mammalian cells for the production of integral membrane proteins.
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spelling pubmed-102164452023-05-27 Screening of Membrane Protein Production by Comparison of Transient Expression in Insect and Mammalian Cells Kaipa, Jagan Mohan Krasnoselska, Ganna Owens, Raymond J. van den Heuvel, Joop Biomolecules Article Membrane proteins are difficult biomolecules to express and purify. In this paper, we compare the small-scale production of six selected eukaryotic integral membrane proteins in insect and mammalian cell expression systems using different techniques for gene delivery. The target proteins were C terminally fused to the green fluorescent marker protein GFP to enable sensitive monitoring. We show that the choice of expression systems makes a considerable difference to the yield and quality of the six selected membrane proteins. Virus-free transient gene expression (TGE) in insect High Five cells combined with solubilization in dodecylmaltoside plus cholesteryl hemisuccinate generated the most homogeneous samples for all six targets. Further, the affinity purification of the solubilized proteins using the Twin-Strep(®) tag improved protein quality in terms of yield and homogeneity compared to His-tag purification. TGE in High Five insect cells offers a fast and economically attractive alternative to the established methods that require either baculovirus construction and the infection of the insect cells or relatively expensive transient gene expression in mammalian cells for the production of integral membrane proteins. MDPI 2023-05-11 /pmc/articles/PMC10216445/ /pubmed/37238687 http://dx.doi.org/10.3390/biom13050817 Text en © 2023 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
Kaipa, Jagan Mohan
Krasnoselska, Ganna
Owens, Raymond J.
van den Heuvel, Joop
Screening of Membrane Protein Production by Comparison of Transient Expression in Insect and Mammalian Cells
title Screening of Membrane Protein Production by Comparison of Transient Expression in Insect and Mammalian Cells
title_full Screening of Membrane Protein Production by Comparison of Transient Expression in Insect and Mammalian Cells
title_fullStr Screening of Membrane Protein Production by Comparison of Transient Expression in Insect and Mammalian Cells
title_full_unstemmed Screening of Membrane Protein Production by Comparison of Transient Expression in Insect and Mammalian Cells
title_short Screening of Membrane Protein Production by Comparison of Transient Expression in Insect and Mammalian Cells
title_sort screening of membrane protein production by comparison of transient expression in insect and mammalian cells
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10216445/
https://www.ncbi.nlm.nih.gov/pubmed/37238687
http://dx.doi.org/10.3390/biom13050817
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