Expression of a chloroplast ATP/ADP transporter in E. coli membranes: Behind the Mistic strategy

CEA, Institut de Biologie Structurale Jean-Pierre Ebel, 41 rue Jules Horowitz, F-38027 Grenoble, France.
Biochimica et Biophysica Acta (Impact Factor: 4.66). 08/2011; 1808(8):2059-66. DOI: 10.1016/j.bbamem.2011.04.011
Source: PubMed


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.

Download full-text


Available from: Annie Frelet-Barrand,
26 Reads
    • "Interestingly, a few years ago the 13- kDa protein Mistic from Bacillus subtilis was described to significantly stimulate heterologous synthesis and activity of hydrophobic proteins when expressed as a fusion with the protein of interest (Roosild et al. 2005; Dvir & Choe 2009). For example, synthesis and functional insertion of the plastidial ATP ⁄ ADP translocase was highly increased due to its expression as a Mistic-fusion (Deniaud et al. 2011). Until now, the in vivo function of Mistic is unclear; however, Mistic is assumed to act like a chaperone that supports protein stability and membrane insertion in the heterologous host. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Membrane transporters set the framework organising the complexity of plant metabolism in cells, tissues and organisms. Their substrate specificity and controlled activity in different cells is a crucial part for plant metabolism to run pathways in concert. Transport proteins catalyse the uptake and exchange of ions, substrates, intermediates, products and cofactors across membranes. Given the large number of metabolites, a wide spectrum of transporters is required. The vast majority of in silico annotated membrane transporters in plant genomes, however, has not yet been functionally characterised. Hence, to understand the metabolic network as a whole, it is important to understand how transporters connect and control the metabolic pathways of plant cells. Heterologous expression and in vitro activity studies of recombinant transport proteins have highly improved their functional analysis in the last two decades. This review provides a comprehensive overview of the recent advances in membrane protein expression and functional characterisation using various host systems and transport assays.
    Plant Biology 05/2012; 14(5). DOI:10.1111/j.1438-8677.2012.00591.x · 2.63 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein.
    PLoS ONE 12/2011; 6(12):e29191. DOI:10.1371/journal.pone.0029191 · 3.23 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Chloroplast ATP/ADP transporters are essential to energy homeostasis in plant cells. However, their molecular mechanism remains poorly understood, primarily due to the difficulty of producing and purifying functional recombinant forms of these transporters. In this work, we describe an expression and purification protocol providing good yields and efficient solubilization of NTT1 protein from Arabidopsis thaliana. By biochemical and biophysical analyses, we identified the best detergent for solubilization and purification of functional proteins, LAPAO. Purified NTT1 was found to accumulate as two independent pools of well folded, stable monomers and dimers. ATP and ADP binding properties were determined, and Pi, a co-substrate of ADP, was confirmed to be essential for nucleotide steady-state transport. Nucleotide binding studies and analysis of NTT1 mutants lead us to suggest the existence of two distinct and probably inter-dependent binding sites. Finally, fusion and deletion experiments demonstrated that the C-terminus of NTT1 is not essential for multimerization, but probably plays a regulatory role, controlling the nucleotide exchange rate. Taken together, these data provide a comprehensive molecular characterization of a chloroplast ATP/ADP transporter.
    PLoS ONE 03/2012; 7(3):e32325. DOI:10.1371/journal.pone.0032325 · 3.23 Impact Factor
Show more