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

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.

Research Highlights
► An ATP/ADP chloroplast transporter can be expressed in mg amounts in fusion with Mistic.
► The fusion with Mistic leads to a low activity ATP/ADP transporter.
► In vivo fusion cleavage delivers a fully active transporter.
► Biophysical characterization shows that Mistic fusion forms large oligomers.
► Well-folded NTT1 mature form can be purified after in vitro fusion cleavage.