title: High yield expression and purification of equilibrative nucleoside transporter 7 (ENT7) from Arabidopsis thaliana.
abstract: BACKGROUND
  Equilibrative nucleoside transporters (ENTs) facilitate the import of nucleosides and their analogs into cells in a bidirectional, non-concentrative manner. However, in contrast to their name, most characterized plant ENTs act in a concentrative manner. A direct characterization of any ENT protein has been hindered due to difficulties in overexpression and obtaining pure recombinant protein.
  
  
  METHODS
  The equilibrative nucleoside transporter 7 from Arabidopsis thaliana (AtENT7) was expressed in Xenopus laevis oocytes to assess mechanism of substrate uptake. Recombinant protein fused to enhanced green fluorescent protein (eGFP) was expressed in Pichia pastoris to characterize its oligomeric state by gel filtration and substrate binding by microscale thermophoresis (MST).
  
  
  RESULTS
  AtENT7 expressed in X. laevis oocytes works as a classic equilibrative transporter. The expression of AtENT7-eGFP in the P. pastoris system yielded milligram amounts of pure protein that exists as stable homodimers. The concentration dependent binding of purine and pyrimidine nucleosides to the purified recombinant protein, assessed by MST, confirmed that AtENT7-eGFP is properly folded. For the first time the binding of nucleobases was observed for AtENT7.
  
  
  CONCLUSIONS
  The availability of pure recombinant AtENT7 will permit detailed kinetic and structural studies of this unique member of the ENT family and, given the functional similarity to mammalian ENTs, will serve as a good model for understanding the structural basis of translocation mechanism for the family.
authors.affiliation: Department of Plant Physiology, University of Kaiserslautern, Erwin-Schrödinger-Straße, D-67663 Kaiserslautern, Germany. Electronic address: girke@rhrk.uni-kl.de.
authors.forename: Christopher
authors.initials: C
authors.lastname: Girke
authors.affiliation: Membrane Protein Disease Research Group, Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada. Electronic address: earutyun@ualberta.ca.
authors.forename: Elena
authors.initials: E
authors.lastname: Arutyunova
authors.affiliation: Membrane Protein Disease Research Group, Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada. Electronic address: masyed@ualberta.ca.
authors.forename: Maria
authors.initials: M
authors.lastname: Syed
authors.affiliation: Department of Plant Physiology, University of Kaiserslautern, Erwin-Schrödinger-Straße, D-67663 Kaiserslautern, Germany. Electronic address: michaela.traub@web.de.
authors.forename: Michaela
authors.initials: M
authors.lastname: Traub
authors.affiliation: Department of Plant Physiology, University of Kaiserslautern, Erwin-Schrödinger-Straße, D-67663 Kaiserslautern, Germany. Electronic address: moehlmann@biologie.uni-kl.de.
authors.forename: Torsten
authors.initials: T
authors.lastname: Möhlmann
authors.affiliation: Membrane Protein Disease Research Group, Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada. Electronic address: joanne.lemieux@ualberta.ca.
authors.forename: M Joanne
authors.initials: MJ
authors.lastname: Lemieux
mesh_terms: D000818:Animals; D017360:Arabidopsis / Q000378:metabolism*; D033705:Equilibrative Nucleoside Transport Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D009865:Oocytes; D011994:Recombinant Proteins / Q000096:biosynthesis*; D014982:Xenopus laevis / Q000235:genetics
chemical_list: D033705:Equilibrative Nucleoside Transport Proteins; D011994:Recombinant Proteins
keywords: Arabidopsis thaliana; ENT7; Equilibrative nucleoside transporter; FSEC; Fluorescence size exclusion chromatography; MST; Membrane transport; Microscale thermophoresis; Nucleobase; Nucleoside; Recombinant expression; Reconstitution