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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

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.

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    • "Mistic is a uniquely hydrophilic membrane protein first identified from Bacillus subtilis and found to be exceptionally efficient in chaperoning the expression of other integral membrane proteins at high yields in Escherichia coli (Deniaud et al., 2011; Roosild et al., 2005; Petrovskaya et al., 2010). It has been proposed that Mistic is capable of autonomously integrating, in a Sec-independent manner, into the lipid bilayer, providing a novel strategy to be explored in L. lactis. "
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    • "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. "
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