Bacterial expression of a eukaryotic membrane protein in fusion to various Mistic orthologs

Mistic, a bacterial membrane-associating protein family, uniquely found in Bacillus species. It enhances expression of eukaryotic membrane proteins at the bacterial membrane. Mistic from B. subtilis (M110), expresses at the Escherichia coli membrane, however its shorter orthologs have been recently shown to be mainly cytoplasmic with varying membrane affinities. Based on that, we hypothesized that the expression level of membrane proteins fused to Mistic is correlated with the degree of membrane association of the particular Mistic protein. We compared expression levels by various Mistic proteins as fusion partners for the Aplysia californica Kv1.1 (aKv1.1) channel as a cargo membrane protein. Mistic from B. atrophaeus (M4), which has the highest membrane association among the shorter orthologs, enhanced expression of the transmembrane domain of aKv1.1 to the highest extent. In contrast, M1, which consists of the 84 C-terminal amino acids of M110 is the most soluble protein and showed the least capacity to express the channel. A chimeric Mistic, constructed with the first α-helix (H1) of M110 N-terminally fused to M4, did not increase the level of expression of aKv1.1 beyond those of either the M110 or the M4 fusions. The channel fused to M110, M4 or the aforementioned H1-M4 chimera, expresses in the highest quantity and quality among Mistic proteins, providing suitable sample for structural studies. Our data support the concept that expression levels of ‘Misticated’ membrane proteins are related to the independent chaperoning character of Mistic via direct membrane association, rather than related to specific sequence-dependent interaction with the E. coli translocon machinery.     

Application of Mistic to improving the expression and membrane integration of histidine kinase receptors from <Emphasis Type="Italic">Escherichia coli</Emphasis>

Integral membrane proteins have become the focus of interest of many laboratories and structural genomics consortia, but their study is hampered by bottlenecks in production, solubilization, purification and crystallization. In our laboratory we have addressed the problem of high-level protein expression in the membrane of Escherichia coli by use of Mistic, a novel Bacillus subtilis protein, as a fusion partner. In this study we examine the effect of Mistic on protein expression and membrane integration levels of members of the E. coli histidine kinase receptor family. We find that Mistic fusion invariably increases the overall yield by targeting the cargo proteins more efficiently to the membrane and may even replace the signal sequence. Mistic fusion methods will likely be instrumental for high-level expression of other integral membrane proteins.     

Mistic: Cellular localization,solution behavior,polymerization, and fibril formation

Mistic represents a family of unique membrane‐associating proteins originally found in Bacillus subtilis (M110). As a fusion partner, it has been shown to assist overexpression of foreign integral membrane proteins in E. coli. We have expressed shorter Mistic homologs from other Bacillus species and surprisingly, unlike M110, found them abundant in the cytoplasm. These Mistic homologs including the corresponding shorter sequence (amino acids 27 through 110 of M110) exist as multimeric assemblies in solution in the absence of detergent. Crystals of Mistic from B. leicheniformis (M2) diffracted to 3.2 Å resolution, indicating that it exists as a multimer in the crystalline state as well. Moreover, we show that although M2 is mostly α‐helical, it tends to polymerize and form fibrils. Such oligomerization could potentially mask the charged surface of the monomeric Mistic to assist membrane integration.     

Mistic's membrane association and its assistance in overexpression of a human GPCR are independent processes

The interaction of the Bacillus subtilis protein Mistic with the bacterial membrane and its role in promoting the overexpression of other membrane proteins are still matters of debate. In this study, we aimed to determine whether individual helical fragments of Mistic are sufficient for its interaction with membranes in vivo and in vitro. To this end, fragments encompassing each of Mistic's helical segments and combinations of them were produced as GFP‐fusions, and their cellular localization was studied in Escherichia coli. Furthermore, peptides corresponding to the four helical fragments were synthesized by solid‐phase peptide synthesis, and their ability to acquire secondary structure in a variety of lipids and detergents was studied by circular dichroism spectroscopy. Both types of experiments demonstrate that the third helical fragment of Mistic interacts only with LDAO micelles but does not partition into lipid bilayers. Interestingly, the other three helices interact with membranes in vivo and in vitro. Nevertheless, all of these short sequences can replace full‐length Mistic as N‐terminal fusions to achieve overexpression of a human G‐protein‐coupled receptor in E. coli, although with different effects on quantity and quality of the protein produced. A bioinformatic analysis of the Mistic family expanded the number of homologs from 4 to 20, including proteins outside the genus Bacillus. This information allowed us to discover a highly conserved Shine‐Dalgarno sequence in the operon mstX‐yugO that is important for downstream translation of the potassium ion channel yugO.     

Identification and bioinformatic analysis of the membrane proteins of synechocystis sp. PCC 6803

Background  

The membranes of Synechocystis sp. PCC 6803 play a central role in photosynthesis, respiration and other important metabolic pathways. Comprehensive identification of the membrane proteins is of importance for a better understanding of the diverse functions of its unique membrane structures. Up to date, approximately 900 known or predicted membrane proteins, consisting 24.5% of Synechocystis sp. PCC 6803 proteome, have been indentified by large-scale proteomic studies.     

Characterization of hypothetical proteins Cpn0146, 0147, 0284 &; 0285 that are predicted to be in the <Emphasis Type="Italic">Chlamydia pneumoniae</Emphasis> inclusion membrane

Background  

Although more than 100 Chlamydia pneumoniae hypothetical proteins have been predicted to be inclusion membrane proteins, only a few have been experimentally demonstrated to be in the inclusion membrane. Using antibodies raised with fusion proteins, we characterized four such hypothetical proteins encoded by two gene clusters (Cpn0146-147 and Cpn0284-285) in the C. pneumoniae genome.     

Cytokinesis is blocked in mammalian cells transfected with Chlamydia trachomatis gene CT223

Background  

The chlamydiae alter many aspects of host cell biology, including the division process, but the molecular biology of these alterations remains poorly characterized. Chlamydial inclusion membrane proteins (Incs) are likely candidates for direct interactions with host cell cytosolic proteins, as they are secreted to the inclusion membrane and exposed to the cytosol. The inc gene CT223 is one of a sequential set of orfs that encode or are predicted to encode Inc proteins. CT223p is localized to the inclusion membrane in all tested C. trachomatis serovars.     

Formation of functional Tat translocases from heterologous components

Background  

The Tat pathway transports folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of plants. In Eschericha coli, Tat transport requires the integral membrane proteins TatA, TatB and TatC. In this study we have tested the ability of tat genes from the eubacterial species Pseudomonas syringae, Streptomyces coelicolor and Aquifex aeolicus, to compensate for the absence of the cognate E. coli tat gene, and thus to form functional Tat translocases with E. coli Tat components.     

Mechanism of protonophores-mediated induction of heat-shock response in Escherichia coli

Background  

Protonophores are the agents that dissipate the proton-motive-force (PMF) across E. coli plasma membrane. As the PMF is known to be an energy source for the translocation of membrane and periplasmic proteins after their initial syntheses in cell cytoplasm, protonophores therefore inhibit the translocation phenomenon. In addition, protonophores also induce heat-shock-like stress response in E. coli cell. In this study, our motivation was to investigate that how the protonophores-mediated phenomena like inhibition of protein translocation and induction of heat-shock proteins in E. coli were correlated.     

Analysis of outer membrane vesicle associated proteins isolated from the plant pathogenic bacterium <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. campestris

Background  

Outer membrane vesicles (OMVs) are released from the outer membrane of many Gram-negative bacteria. These extracellular compartments are known to transport compounds involved in cell-cell signalling as well as virulence associated proteins, e.g. the cytolysine from enterotoxic E. coli.     

Immunoproteomic analysis of outer membrane proteins and extracellular proteins of Actinobacillus pleuropneumoniae JL03 serotype 3

Background  

Actinobacillus pleuropneumoniae is the causative agent of porcine contagious pleuropneumonia, a highly contagious respiratory infection in pigs, and all the 15 serotypes are able to cause disease. Current vaccines including subunit vaccines could not provide satisfactory protection against A. pleuropneumoniae. In this study, the immunoproteomic approach was applied to the analysis of extracellular and outer membrane proteins of A. pleuropneumoniae JL03 serotype 3 for the identification of novel immunogenic proteins for A. pleuropneumoniae.     

Protein cleavage strategies for an improved analysis of the membrane proteome

Background  

Membrane proteins still remain elusive in proteomic studies. This is in part due to the distribution of the amino acids lysine and arginine, which are less frequent in integral membrane proteins and almost absent in transmembrane helices. As these amino acids are cleavage targets for the commonly used protease trypsin, alternative cleavage conditions, which should improve membrane protein analysis, were tested by in silico digestion for the three organisms Saccharomyces cerevisiae, Halobacterium sp. NRC-1, and Corynebacterium glutamicum as hallmarks for eukaryotes, archea and eubacteria.     

A proteomic study of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>AAA+ protease FtsH

Background  

The influence of the membrane-bound AAA+ protease FtsH on membrane and cytoplasmic proteins of Corynebacterium glutamicum was investigated in this study. For the analysis of the membrane fraction, anion exchange chromatography was combined with SDS-PAGE, while the cytoplasmic protein fraction was studied by conventional two-dimensional gel electrophoresis.     

Vesicle-independent extracellular release of a proinflammatory outer membrane lipoprotein in free-soluble form

Background  

Aggregatibacter actinomycetemcomitans is an oral bacterium associated with aggressively progressing periodontitis. Extracellular release of bacterial outer membrane proteins has been suggested to mainly occur via outer membrane vesicles. This study investigated the presence and conservation of peptidoglycan-associated lipoprotein (AaPAL) among A. actinomycetemcomitans strains, the immunostimulatory effect of AaPAL, and whether live cells release this structural outer membrane lipoprotein in free-soluble form independent of vesicles.     

A method for protein extraction from different subcellular fractions of laticifer latex in <Emphasis Type="Italic">Hevea brasiliensis</Emphasis> compatible with 2-DE and MS

Background  

Proteomic analysis of laticifer latex in Hevea brasiliensis has been received more significant attentions. However, the sticky and viscous characteristic of rubber latex as cytoplasm of laticifer cells and the complication of laticifer latex membrane systems has made it challenge to isolate high-quality proteins for 2-DE and MS.     

Chlamydial development is blocked in host cells transfected with <Emphasis Type="Italic">Chlamydophila caviae incA</Emphasis>

Background  

Chlamydiae produce a set of proteins, termed Inc proteins, that are localized to the inclusion membrane and exposed to the host cell cytosol. Little information exists regarding the interaction of Inc proteins with the eukaryotic cell. To examine these interactions, Vaccinia virus vectors and mammalian plasmid-based systems were used to express inc genes in mammalian cells.     

Adaptation of <Emphasis Type="Italic">Salmonella enterica</Emphasis> Hadar under static magnetic field: effects on outer membrane protein pattern

Background  

Salmonella enterica serovar Hadar (S. Hadar) is a highly prevalent foodborne pathogen and therefore a major cause of human gastroenteritis worldwide. Outer membrane proteins whose production is often regulated by environmental conditions also play important roles in the adaptability of bacterial pathogens to various environments.     

Heterologous expression of membrane proteins: choosing the appropriate host

Background

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.

Methodology/Principal Findings

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.

Conclusions/Significance

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.     

Towards the development of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> as a cell factory for membrane proteins and protein complexes

Background  

The Gram-positive bacterium Bacillus subtilis is an important producer of high quality industrial enzymes and a few eukaryotic proteins. Most of these proteins are secreted into the growth medium, but successful examples of cytoplasmic protein production are also known. Therefore, one may anticipate that the high protein production potential of B. subtilis can be exploited for protein complexes and membrane proteins to facilitate their functional and structural analysis. The high quality of proteins produced with B. subtilis results from the action of cellular quality control systems that efficiently remove misfolded or incompletely synthesized proteins. Paradoxically, cellular quality control systems also represent bottlenecks for the production of various heterologous proteins at significant concentrations.     

Varicose: a MAGUK required for the maturation and function of <Emphasis Type="Italic">Drosophila</Emphasis> septate junctions

Background  

Scaffolding proteins belonging to the membrane associated guanylate kinase (MAGUK) superfamily function as adapters linking cytoplasmic and cell surface proteins to the cytoskeleton to regulate cell-cell adhesion, cell-cell communication and signal transduction. We characterize here a Drosophila MAGUK member, Varicose (Vari), the homologue of vertebrate scaffolding protein PALS2.