An efficient strategy for small-scale screening and production of archaeal membrane transport proteins in Escherichia coli.
<h4>Background</h4>Membrane proteins play a key role in many fundamental cellular processes such as transport of nutrients, sensing of environmental signals and energy transduction, and account for over 50% of all known drug targets. Despite their importance, structural and functional ch...
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2013-01-01
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| Online Access: | https://doi.org/10.1371/journal.pone.0076913 |
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| author | Pikyee Ma Filipa Varela Malgorzata Magoch Ana Rita Silva Ana Lúcia Rosário José Brito Tânia Filipa Oliveira Przemyslaw Nogly Miguel Pessanha Meike Stelter Arnulf Kletzin Peter J F Henderson Margarida Archer |
| author_facet | Pikyee Ma Filipa Varela Malgorzata Magoch Ana Rita Silva Ana Lúcia Rosário José Brito Tânia Filipa Oliveira Przemyslaw Nogly Miguel Pessanha Meike Stelter Arnulf Kletzin Peter J F Henderson Margarida Archer |
| author_sort | Pikyee Ma |
| collection | DOAJ |
| description | <h4>Background</h4>Membrane proteins play a key role in many fundamental cellular processes such as transport of nutrients, sensing of environmental signals and energy transduction, and account for over 50% of all known drug targets. Despite their importance, structural and functional characterisation of membrane proteins still remains a challenge, partially due to the difficulties in recombinant expression and purification. Therefore the need for development of efficient methods for heterologous production is essential.<h4>Methodology/principal findings</h4>Fifteen integral membrane transport proteins from Archaea were selected as test targets, chosen to represent two superfamilies widespread in all organisms known as the Major Facilitator Superfamily (MFS) and the 5-Helix Inverted Repeat Transporter superfamily (5HIRT). These proteins typically have eleven to twelve predicted transmembrane helices and are putative transporters for sugar, metabolite, nucleobase, vitamin or neurotransmitter. They include a wide range of examples from the following families: Metabolite-H(+)-symporter; Sugar Porter; Nucleobase-Cation-Symporter-1; Nucleobase-Cation-Symporter-2; and neurotransmitter-sodium-symporter. Overproduction of transporters was evaluated with three vectors (pTTQ18, pET52b, pWarf) and two Escherichia coli strains (BL21 Star and C43 (DE3)). Thirteen transporter genes were successfully expressed; only two did not express in any of the tested vector-strain combinations. Initial trials showed that seven transporters could be purified and six of these yielded quantities of ≥ 0.4 mg per litre suitable for functional and structural studies. Size-exclusion chromatography confirmed that two purified transporters were almost homogeneous while four others were shown to be non-aggregating, indicating that they are ready for up-scale production and crystallisation trials.<h4>Conclusions/significance</h4>Here, we describe an efficient strategy for heterologous production of membrane transport proteins in E. coli. Small-volume cultures (10 mL) produced sufficient amount of proteins to assess their purity and aggregation state. The methods described in this work are simple to implement and can be easily applied to many more membrane proteins. |
| format | Article |
| id | doaj-art-96885eedb2ff495cab7fd67f03de5d31 |
| institution | OA Journals |
| issn | 1932-6203 |
| language | English |
| publishDate | 2013-01-01 |
| publisher | Public Library of Science (PLoS) |
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| series | PLoS ONE |
| spelling | doaj-art-96885eedb2ff495cab7fd67f03de5d312025-08-20T02:22:37ZengPublic Library of Science (PLoS)PLoS ONE1932-62032013-01-01810e7691310.1371/journal.pone.0076913An efficient strategy for small-scale screening and production of archaeal membrane transport proteins in Escherichia coli.Pikyee MaFilipa VarelaMalgorzata MagochAna Rita SilvaAna Lúcia RosárioJosé BritoTânia Filipa OliveiraPrzemyslaw NoglyMiguel PessanhaMeike StelterArnulf KletzinPeter J F HendersonMargarida Archer<h4>Background</h4>Membrane proteins play a key role in many fundamental cellular processes such as transport of nutrients, sensing of environmental signals and energy transduction, and account for over 50% of all known drug targets. Despite their importance, structural and functional characterisation of membrane proteins still remains a challenge, partially due to the difficulties in recombinant expression and purification. Therefore the need for development of efficient methods for heterologous production is essential.<h4>Methodology/principal findings</h4>Fifteen integral membrane transport proteins from Archaea were selected as test targets, chosen to represent two superfamilies widespread in all organisms known as the Major Facilitator Superfamily (MFS) and the 5-Helix Inverted Repeat Transporter superfamily (5HIRT). These proteins typically have eleven to twelve predicted transmembrane helices and are putative transporters for sugar, metabolite, nucleobase, vitamin or neurotransmitter. They include a wide range of examples from the following families: Metabolite-H(+)-symporter; Sugar Porter; Nucleobase-Cation-Symporter-1; Nucleobase-Cation-Symporter-2; and neurotransmitter-sodium-symporter. Overproduction of transporters was evaluated with three vectors (pTTQ18, pET52b, pWarf) and two Escherichia coli strains (BL21 Star and C43 (DE3)). Thirteen transporter genes were successfully expressed; only two did not express in any of the tested vector-strain combinations. Initial trials showed that seven transporters could be purified and six of these yielded quantities of ≥ 0.4 mg per litre suitable for functional and structural studies. Size-exclusion chromatography confirmed that two purified transporters were almost homogeneous while four others were shown to be non-aggregating, indicating that they are ready for up-scale production and crystallisation trials.<h4>Conclusions/significance</h4>Here, we describe an efficient strategy for heterologous production of membrane transport proteins in E. coli. Small-volume cultures (10 mL) produced sufficient amount of proteins to assess their purity and aggregation state. The methods described in this work are simple to implement and can be easily applied to many more membrane proteins.https://doi.org/10.1371/journal.pone.0076913 |
| spellingShingle | Pikyee Ma Filipa Varela Malgorzata Magoch Ana Rita Silva Ana Lúcia Rosário José Brito Tânia Filipa Oliveira Przemyslaw Nogly Miguel Pessanha Meike Stelter Arnulf Kletzin Peter J F Henderson Margarida Archer An efficient strategy for small-scale screening and production of archaeal membrane transport proteins in Escherichia coli. PLoS ONE |
| title | An efficient strategy for small-scale screening and production of archaeal membrane transport proteins in Escherichia coli. |
| title_full | An efficient strategy for small-scale screening and production of archaeal membrane transport proteins in Escherichia coli. |
| title_fullStr | An efficient strategy for small-scale screening and production of archaeal membrane transport proteins in Escherichia coli. |
| title_full_unstemmed | An efficient strategy for small-scale screening and production of archaeal membrane transport proteins in Escherichia coli. |
| title_short | An efficient strategy for small-scale screening and production of archaeal membrane transport proteins in Escherichia coli. |
| title_sort | efficient strategy for small scale screening and production of archaeal membrane transport proteins in escherichia coli |
| url | https://doi.org/10.1371/journal.pone.0076913 |
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