title: An efficient strategy for small-scale screening and production of archaeal membrane transport proteins in Escherichia coli.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Instituto de Tecnologia Quίmica e Biolόgica, Universidade Nova de Lisboa, Oeiras, Portugal.
authors.forename: Pikyee
authors.initials: P
authors.lastname: Ma
authors.forename: Filipa
authors.initials: F
authors.lastname: Varela
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Magoch
authors.forename: Ana Rita
authors.initials: AR
authors.lastname: Silva
authors.forename: Ana Lúcia
authors.initials: AL
authors.lastname: Rosário
authors.forename: José
authors.initials: J
authors.lastname: Brito
authors.forename: Tânia Filipa
authors.initials: TF
authors.lastname: Oliveira
authors.forename: Przemyslaw
authors.initials: P
authors.lastname: Nogly
authors.forename: Miguel
authors.initials: M
authors.lastname: Pessanha
authors.forename: Meike
authors.initials: M
authors.lastname: Stelter
authors.forename: Arnulf
authors.initials: A
authors.lastname: Kletzin
authors.forename: Peter J F
authors.initials: PJ
authors.lastname: Henderson
authors.forename: Margarida
authors.initials: M
authors.lastname: Archer
mesh_terms: D001105:Archaea / Q000235:genetics; D019843:Archaeal Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004926:Escherichia coli; D015870:Gene Expression; D026901:Membrane Transport Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
chemical_list: D019843:Archaeal Proteins; D026901:Membrane Transport Proteins; D011994:Recombinant Proteins