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An efficient strategy for small-scale screening and production of archaeal membrane transport proteins in Escherichia coli.

التفاصيل البيبلوغرافية
العنوان: An efficient strategy for small-scale screening and production of archaeal membrane transport proteins in Escherichia coli.
المؤلفون: 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
المصدر: PLoS ONE, Vol 8, Iss 10, p e76913 (2013)
بيانات النشر: Public Library of Science (PLoS), 2013.
سنة النشر: 2013
المجموعة: LCC:Medicine
LCC:Science
مصطلحات موضوعية: Medicine, Science
الوصف: 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.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.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.
نوع الوثيقة: article
وصف الملف: electronic resource
اللغة: English
تدمد: 1932-6203
Relation: http://europepmc.org/articles/PMC3838208?pdf=render; https://doaj.org/toc/1932-6203
DOI: 10.1371/journal.pone.0076913
URL الوصول: https://doaj.org/article/96885eedb2ff495cab7fd67f03de5d31
رقم الأكسشن: edsdoj.96885eedb2ff495cab7fd67f03de5d31
قاعدة البيانات: Directory of Open Access Journals
الوصف
تدمد:19326203
DOI:10.1371/journal.pone.0076913