title: Expression of a chloroplast ATP/ADP transporter in E. coli membranes: behind the Mistic strategy.
abstract: Eukaryotic membrane protein expression is still a major bottleneck for structural studies. Production in E. coli often leads to low expression level and/or aggregated proteins. In the last decade, strategies relying on new fusion protein expression revealed promising results. Fusion with the amphipatic Mistic protein has been described to favor expression in E. coli membranes. Although, this approach has already been reported for a few membrane proteins, little is known about the activity of the fused proteins. We used this strategy and obtained high expression levels of a chloroplast ATP/ADP transporter from A. thaliana (NTT1) and characterized its transport properties. NTT1 fused to Mistic has a very low transport activity which can be recovered after in vivo Mistic fusion cleavage. Moreover, detailed molecular characterization of purified NTT1 mature form, NTT1 fused to Mistic or NTT1 cleaved-off from this fusion highlights the correct fold of the latter one. Therefore, considering the higher quantity of purified NTT1 mature form obtained via the Mistic fusion approach, this is a valuable strategy for obtaining quantities of pure and active proteins that are adequate for structural studies.
authors.affiliation: CEA, Institut de Biologie Structurale Jean-Pierre Ebel, 41 rue Jules Horowitz, F-38027 Grenoble, France.
authors.forename: Aurélien
authors.initials: A
authors.lastname: Deniaud
authors.forename: Florent
authors.initials: F
authors.lastname: Bernaudat
authors.forename: Annie
authors.initials: A
authors.lastname: Frelet-Barrand
authors.forename: Céline
authors.initials: C
authors.lastname: Juillan-Binard
authors.forename: Thierry
authors.initials: T
authors.lastname: Vernet
authors.forename: Norbert
authors.initials: N
authors.lastname: Rolland
authors.forename: Eva
authors.initials: E
authors.lastname: Pebay-Peyroula
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D029845:Amphibian Proteins / Q000096:biosynthesis* / Q000235:genetics; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism*; D002736:Chloroplasts / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D033704:Nucleotide Transport Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D010447:Peptide Hydrolases / Q000378:metabolism; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D013329:Structure-Activity Relationship
chemical_list: D029845:Amphibian Proteins; D029681:Arabidopsis Proteins; D033704:Nucleotide Transport Proteins; D011993:Recombinant Fusion Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D010447:Peptide Hydrolases