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.

Oligomeric Status and Nucleotide Binding Properties of the Plastid ATP/ADP Transporter 1: Toward a Molecular Understanding of the Transport Mechanism

“…The main construct used in this study is described in detail in [6] and corresponds to Uniprot sequence number Q39002 lacking the first 79 residues corresponding to the N-terminal transit peptide, but containing an N-terminal His-tag and a C-terminal StreptagII . Mutants K155E, K155R, E245K, K527E, K527R and the C-terminal deletion were generated with the primers listed in table S1, using the Quick Change Site Directed Mutagenesis kit (Agilent Technologies).…”

“…Producing an active form of NTT1 in heterologous systems thus requires expression of the corresponding mature protein. In the specific case of NTT1 from Arabidopsis thaliana , three expression systems allowed the production of functional NTT1: E. coli [6], L. lactis [7] and A. thaliana .…”

“…These recent reports also provided the first descriptions of purification procedures for recombinant NTT1 proteins produced in bacteria [3], [9]. Using recombinant NTT1 produced either as a mature form of the protein or fused with Mistic [6], we recently got first insights into the oligomeric status of this transporter. In this study, the biochemical properties of this transporter were further characterized.…”

Membrane Protein Production for Structural Analysis

“…This strategy was used to express the chloroplast ATP/ADP transporter from Arabidopsis thaliana (NTT1) and characterize its transport properties (Deniaud et al 2011). NTT1 fused to Mistic has a very low transport activity, which can be recovered after in vivo Mistic fusion cleavage.…”

Improved membrane protein expression in Lactococcus lactis by fusion to Mistic

“…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.…”