A Versatile Strategy for Production of Membrane Proteins with Diverse Topologies: Application to Investigation of Bacterial Homologues of Human Divalent Metal Ion and Nucleoside Transporters

Membrane proteins play key roles in many biological processes, from acquisition of nutrients to neurotransmission, and are targets for more than 50% of current therapeutic drugs. However, their investigation is hampered by difficulties in their production and purification on a scale suitable for structural studies. In particular, the nature and location of affinity tags introduced for the purification of recombinant membrane proteins can greatly influence their expression levels by affecting their membrane insertion. The extent of such effects typically depends on the transmembrane topologies of the proteins, which for proteins of unknown structure are usually uncertain. For example, attachment of oligohistidine tags to the periplasmic termini of membrane proteins often interferes with folding and drastically impairs expression in Escherichia coli. To circumvent this problem we have employed a novel strategy to enable the rapid production of constructs bearing a range of different affinity tags compatible with either cytoplasmic or periplasmic attachment. Tags include conventional oligohistidine tags compatible with cytoplasmic attachment and, for attachment to proteins with a periplasmic terminus, either tandem Strep-tag II sequences or oligohistidine tags fused to maltose binding protein and a signal sequence. Inclusion of cleavage sites for TEV or HRV-3C protease enables tag removal prior to crystallisation trials or a second step of purification. Together with the use of bioinformatic approaches to identify members of membrane protein families with topologies favourable to cytoplasmic tagging, this has enabled us to express and purify multiple bacterial membrane transporters. To illustrate this strategy, we describe here its use to purify bacterial homologues of human membrane proteins from the Nramp and ZIP families of divalent metal cation transporters and from the concentrative nucleoside transporter family. The proteins are expressed in E. coli in a correctly folded, functional state and can be purified in amounts suitable for structural investigations.     

细菌脲酶蛋白复合物UreABC的表达与纯化

细菌脲酶能分解尿素为氨,在瘤胃尿素氮代谢中发挥重要作用.为了表达纯化并研究蛋白脲酶复合物UreABC,通过PCR扩增脲酶基因簇的结构基因ureA、ureB、ureC,将ureB构建在含有N端His标签的pet28a+载体,ureA、ureC基因共同构建在含有N端His标签的表达载体pETDuet-1,经酶切及测序鉴定得...     

Consequences of the overexpression of a eukaryotic membrane protein, the human KDEL receptor, in Escherichia coli

Escherichia coli is the most widely used host for producing membrane proteins. Thus far, to study the consequences of membrane protein overexpression in E. coli, we have focussed on prokaryotic membrane proteins as overexpression targets. Their overexpression results in the saturation of the Sec translocon, which is a protein-conducting channel in the cytoplasmic membrane that mediates both protein translocation and insertion. Saturation of the Sec translocon leads to (i) protein misfolding/aggregation in the cytoplasm, (ii) impaired respiration, and (iii) activation of the Arc response, which leads to inefficient ATP production and the formation of acetate. The overexpression yields of eukaryotic membrane proteins in E. coli are usually much lower than those of prokaryotic ones. This may be due to differences between the consequences of the overexpression of prokaryotic and eukaryotic membrane proteins in E. coli. Therefore, we have now also studied in detail how the overexpression of a eukaryotic membrane protein, the human KDEL receptor, affects E. coli. Surprisingly, the consequences of the overexpression of a prokaryotic and a eukaryotic membrane protein are very similar. Strain engineering and likely also protein engineering can be used to remedy the saturation of the Sec translocon upon overexpression of both prokaryotic and eukaryotic membrane proteins in E. coli.     

A novel chimeric MOMP antigen expressed in Escherichia coli, Arabidopsis thaliana, and Daucus carota as a potential Chlamydia trachomatis vaccine candidate

The major outer membrane protein (MOMP) of Chlamydia trachomatis is a highly antigenic and hydrophobic transmembrane protein. Our attempts to express the full-length protein in a soluble form in Escherichia coli and in transgenic plants failed. A chimeric gene construct of C. trachomatis serovar E MOMP was designed in order to increase solubility of the MOMP protein but with retained antigenicity. The designed construct was successfully expressed in E. coli, in Arabidopsis thaliana, and in Daucus carota. The chimeric MOMP expressed in and purified from E. coli was used as antigen for production of antibodies in rabbits. The anti-chimeric MOMP antibodies recognized the corresponding protein in both E. coli and in transgenic plants, as well as in inactivated C. trachomatis elementary bodies. Transgenic Arabidopsis and carrots were characterized for the number of MOMP chimeric genetic inserts and for protein expression. Stable integration of the transgene and the corresponding protein expression were demonstrated in Arabidopsis plants over at least six generations. Transgenic carrots showed a high level of expression of the chimeric MOMP – up to 3% of TSP.     

Large‐scale identification of membrane proteins with properties favorable for crystallization

Membrane protein crystallography is notoriously difficult due to challenges in protein expression and issues of degradation and structural stability. We have developed a novel method for large‐scale screening of native sources for integral membrane proteins that have intrinsic biochemical properties favorable for crystallization. Highly expressed membrane proteins that are thermally stable and nonaggregating in detergent solutions were identified by mass spectrometry from Escherichia coli, Saccharomyces cerevisiae, and Sus scrofa cerebrum. Many of the membrane proteins identified had been crystallized previously, supporting the promise of the approach. Most identified proteins have known functions and include high‐value targets such as transporters and ATPases. To validate the method, we recombinantly expressed and purified the yeast protein, Yop1, which is responsible for endoplasmic reticulum curvature. We demonstrate that Yop1 can be purified with the detergent dodecylmaltoside without aggregating.     

Expression and secretion of proteins in E. coli

This review outlines approaches to the cloning and expression of proteins in Escherichia coli. The expression vectors described here (pIN-III derivatives) utilize the strong lipoprotein promoter, which is controlled by the lac-UV5 promoter-operator. These vectors provide the means for targeting a protein to any of the four subcellular compartments of the bacterial cell: cytoplasm, cytoplasmic membrane, periplasm, and outer membrane. Of particular importance is that secretion of proteins into the E. coli periplasm (using the OmpA signal peptide) is applicable for the production of both prokaryotic and eukaryotic proteins thereby enhancing protein activity and stability.     

Rapid automated detergent screening for the solubilization and purification of membrane proteins and complexes

Membrane proteins constitute about one third of proteins encoded by all genomes, but only a small percentage have their structures deposited in the Protein Data Bank. One bottleneck in the pipeline from expression to structure determination is the identification of detergents that maintain the protein in a soluble, stable, and active state. Here, we describe a small‐scale automated procedure to easily and rapidly screen detergents for the solubilization and purification of membrane proteins, to perform detergent exchange, or to identify conditions preserving protein interactions in complexes. Hundreds of conditions can be tested in a few hours to select detergents that keep proteins folded and nonaggregated, from single membrane preparations of cells overexpressing the protein(s) of interest. Thirty‐one prokaryotic, eukaryotic, and viral membrane proteins were analyzed by our small‐scale procedure to identify the best‐associated detergents. Examples of results obtained with a bitopic and multitopic membrane proteins and membrane protein complexes are presented in more detail. DDM, DM, DMNG, TritonX‐100, LAPAO, and Fos‐12 appeared effective for successful membrane solubilization and protein purification of most selected targets. Eukaryotic proteins are in general more difficult to extract and purify from Escherichia coli membranes than prokaryotic proteins. The protocol has been developed for His‐tagged proteins, but can readily be adapted to other affinity tags by adjusting the chromatography resin and the buffer composition.     

Pseudomonas aeruginosa outer membrane protein F produced inEscherichia coli retains vaccine efficacy

Pseudomonas aeruginosa outer membrane protein F was purified by extraction from polyacrylamide gels of cell envelope proteins of anEscherichia coli strain expressing the cloned gene for protein F. Antisera directed against protein F purified fromP. aeruginosa PAO1 reacted with thisE. coli strain by immunofluorescence assay and immunoblotting, whereas these antisera were nonreactive withE. coli strains lacking thePseudomonas protein F gene. The protein F purified from thisE. coli strain was used to immunize mice by intramuscular injection of 10 µg of protein F preparation on days 1 and 14, followed by burn and challenge of the mice on day 28. As compared with control mice immunized withE. coli K-12 lipopolysaccharide, immunization with theE. coli-derived protein F afforded significant protection against subsequent challenge with heterologous Fisher-Devlin immunotype 5 and 6 strains ofP. aeruginosa. Antisera from mice immunized with theE. coli-derived protein F reacted at bands corresponding to protein F and 2-mercaptoethanol-modified protein F upon immunoblotting against cell envelope proteins of the PAO1, immunotype 5, and immunotype 6 strains ofP. aeruginosa and theE. coli strain containing the cloned F gene, but failed to react at these sites in anE. coli strain lacking the F gene. These data demonstrate thatP. aeruginosa protein F produced inE. coli through genetic engineering techniques retains its vaccine efficacy in the complete absence of anyP. aeruginosa lipopolysaccharide.     

改造大肠杆菌卟啉代谢途径对重组过氧化物酶活性的影响

【目的】原核表达某些需辅因子的外源蛋白时往往酶活偏低,为提高酶活和减少外加辅因子的成本,我们尝试在大肠杆菌中表达外源过氧化氢-过氧化物酶的同时提高大肠杆菌中与该酶辅因子相关的合成代谢。【方法】本研究克隆了中度嗜盐菌Halomonas elongata DSM2581的过氧化氢-过氧化物酶CAT-POD(catalase-peroxidase)编码基因kat G的ORF,构建原核表达载体p ET28a-kat G,实现了CAT-POD在大肠杆菌中的重组表达。由于CAT-POD活性依赖其活性中心血红素,而血卟啉是血红素的骨架,通过构建原核表达载体p UC19-tac-hem A,将编码5-氨基乙酰丙酸合成酶的hem A基因在大肠杆菌中过量表达,提高卟啉的含量,从而提高重组蛋白CAT-POD的酶活。【结果】最终的CAT酶活达到了377 U/m L,为对照组的7.5倍。【结论】本研究为工业生产高活性CAT-POD提供了有效的方案,也为体外重组表达含辅因子的蛋白提供可借鉴的思路。     

Homology between Drosophila melanogaster and Escherichia coli ribosomal proteins

Summary Antibodies raised against D. melanogaster ribosomal proteins were used to examine possible structural relationships between eukaryotic and prokaryotic ribosomal proteins. The antisera were raised against either groups of ribosomal proteins or purified individual ribosomal proteins from D. melanogaster. The specificity of each antiserum was confirmed and the identity of the homologous E. coli ribosomal protein was determined by immunochemical methods. Immuno-overlay assays indicated that the antiserum against the D. melanogaster small subunit protein S14 (anti-S14) was highly specific for protein S14. In addition, anti-S14 showed a cross-reaction with total E. coli ribosomal proteins in Ouchterlony double immunodiffusion assays and with only E. coli protein S6 in immuno-overlay assays. From these and other experiments with adsorption of anti-S14 with individual purified proteins, the E. coli protein homologous to the D. melanogaster protein S14 was established as protein S6.     

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.     

Construction of Escherichia coli dnaK-deletion mutant infected by lambdaDE3 for overexpression and purification of recombinant GrpE proteins

Escherichia coli is widely employed to produce recombinant proteins because this microorganism is simple to manipulate, inexpensive to culture, and of short duration to produce a recombinant protein. However, contamination of molecular chaperone DnaK during purification of the recombinant protein is sometimes a problem, since DnaK sometimes has a negative effect on subsequent experiments. Previously, several efforts have been done to remove the DnaK contaminants by several sequential chromatography or washing with some expensive chemicals such as ATP. Here, we developed a simple and inexpensive method to express and purify recombinant proteins based on an E. coli dnaK-deletion mutant. The E. coli ΔdnaK52 mutant was infected by λDE3 phage to overexpress desired recombinant proteins under the control of T7 promoter. Using this host cell, recombinant hexa histidine-tag fused GrpE, which is well known as a co-chaperone for DnaK and to strongly interact with DnaK, was overexpressed and purified by one-step nickel affinity chromatography. As a result, highly purified recombinant GrpE was obtained without washing with ATP. The purified recombinant GrpE showed a folded secondary structure and a dimeric structure as previous findings. In vitro ATPase activity assay and luciferase-refolding activity assay demonstrated that the recombinant GrpE worked together with DnaK. Thus, this developed method would be rapid and useful for expression and purification of recombinant proteins which is difficult to remove DnaK contaminants.     

Structures of the OmpF porin crystallized in the presence of foscholine‐12

The endogenous Escherichia coli porin OmpF was crystallized as an accidental by‐product of our efforts to express, purify, and crystallize the E. coli integral membrane protein KdpD in the presence of foscholine‐12 (FC12). FC12 is widely used in membrane protein studies, but no crystal structure of a protein that was both purified and crystallized with this detergent has been reported in the Protein Data Bank. Crystallization screening for KdpD yielded two different crystals of contaminating protein OmpF. Here, we report two OmpF structures, the first membrane protein crystal structures for which extraction, purification, and crystallization were done exclusively with FC12. The first structure was refined in space group P21 with cell parameters a = 136.7 Å, b = 210.5 Å, c = 137 Å, and β = 100.5°, and the resolution of 3.8 Å. The second structure was solved at the resolution of 4.4 Å and was refined in the P321 space group, with unit cell parameters a = 215.5 Å, b = 215.5 Å, c = 137.5 Å, and γ = 120°. Both crystal forms show novel crystal packing, in which the building block is a tetrahedral arrangement of four trimers. Additionally, we discuss the use of FC12 for membrane protein crystallization and structure determination, as well as the problem of the OmpF contamination for membrane proteins overexpressed in E. coli.     

Binding of the S7 Protein with Fragment 926–986/1219–1393 of the 16S rRNA As a Key Step in the Assembly of the Small Subunit of Prokaryotic Ribosomes

Both structural and thermodynamic studies are necessary to understand the ribosome assembly. An initial step was made in studying the interaction between a 16S rRNA fragment and S7, a key protein in assembling the prokaryotic ribosome small subunit. The apparent dissociation constant was obtained for complexes of recombinant Escherichia coliandThermus thermophilusS7 with a fragment of the 3" domain of the E. coli16S rRNA. Both proteins showed high rRNA-binding activity, which was not observed earlier. Since RNA and proteins are conformationally labile, their folding must be considered to correctly describe the RNA–protein interactions.     

Characterization,Identification, and Cloning of the S-Layer Protein from <Emphasis Type="Italic">Cytophaga</Emphasis> sp.

We characterized, identified, and cloned a major protein which comprised 16% of the total proteins from Cytophaga sp. cell lysate. After French pressing, the fraction of cell envelope was treated with 0.2% Triton X-100 to remove cell membranes. Subsequent SDS-PAGE analysis of the Triton X-100-insoluble cell wall revealed a protein of 120 kDa with a pI of 5.4, which was identified by gold immunostaining as the surface (S)-layer protein of this soil bacterium. The nucleotide sequence of the cloned S-layer protein gene (slp) encoding this protein consisted of 3144 nucleotides with an ORF for 1047 amino acids, which included a typical 32-amino acid leader peptide sequence. Amino acid sequence alignment revealed 29–48% similarity between this protein and the S-layer proteins from other prokaryotic organisms. The 120-kDa protein from the Cytophaga sp. cell lysate has been characterized as a member of the S-layer proteins, and the slp gene was cloned and expressed in Escherichia coli. E. coli harboring the plasmid containing the 600- or 800-bp DNA fragment upstream of the initiation codon of the slp gene, in the presence of the reporter gene rsda (raw starch digesting amylase), showed amylase activity in starch containing plate. The putative promoter region of slp located 600 bp upstream of the initiation codon might be used for foreign gene expression.     

Structural genomics target selection for the New York consortium on membrane protein structure

The New York Consortium on Membrane Protein Structure (NYCOMPS), a part of the Protein Structure Initiative (PSI) in the USA, has as its mission to establish a high-throughput pipeline for determination of novel integral membrane protein structures. Here we describe our current target selection protocol, which applies structural genomics approaches informed by the collective experience of our team of investigators. We first extract all annotated proteins from our reagent genomes, i.e. the 96 fully sequenced prokaryotic genomes from which we clone DNA. We filter this initial pool of sequences and obtain a list of valid targets. NYCOMPS defines valid targets as those that, among other features, have at least two predicted transmembrane helices, no predicted long disordered regions and, except for community nominated targets, no significant sequence similarity in the predicted transmembrane region to any known protein structure. Proteins that feed our experimental pipeline are selected by defining a protein seed and searching the set of all valid targets for proteins that are likely to have a transmembrane region structurally similar to that of the seed. We require sequence similarity aligning at least half of the predicted transmembrane region of seed and target. Seeds are selected according to their feasibility and/or biological interest, and they include both centrally selected targets and community nominated targets. As of December 2008, over 6,000 targets have been selected and are currently being processed by the experimental pipeline. We discuss how our target list may impact structural coverage of the membrane protein space.     

Protein exporter function and in vitro ATPase activity are correlated in ABC-domain mutants of HlyB

The Escherichia coli toxin exporter HlyB comprises an integral membrane domain fused to a cytoplasmic domain of the ATP-binding casette (ABC) super-family, and it directs translocation of the 110kDa haemolysin protein out of the bacterial cell without using an N-terminal secretion signal peptide. We have exploited the ability to purify the soluble HlyB ABC domain as a fusion with glutathione S-transferase to obtain a direct correlation of the in vivo export of protein by HlyB with the degree of ATP binding and hydrolysis measured in vitro. Mutations in residues that are invariant or highly conserved in the ATP-binding fold and glycine-rich linker peptide of prokaryotic and eukaryotic ABC transporters caused a complete less of both HlyB exporter function and ATPase activity in proteins still able to bind ATP effectively and undergo ATP-induced conformational change. Mutation of less-conserved residues caused reduced export and ATP hydrolysis, but not ATP binding, whereas substitutions of poorly conserved residues did not impair activity either in vivo or in vitro. The data show that protein export by HlyB has an absolute requirement for the hydrolysis of ATP bound by its cytoplasmic domain and indicate that comparable mutations that disable other prokaryotic and eukaryotic ABC transporters also cause a specific loss of enzymatic activity.     

Enhanced Expression of Rabies Virus Surface G-Protein in Escherichia coli using SUMO Fusion

Fusion systems are known to increase the expression of difficult to express recombinant proteins in soluble form to facilitate their purification. Rabies glycoprotein was also tough to express at sufficient level in soluble form in both E. coli and plant. The present work was aimed to over-express and purify this membrane protein from soluble extract of E. coli. Fusion of Small Ubiqutin like Modifier (SUMO) with rabies glycoprotein increased ~1.5 fold higher expression and ~3.0 fold solubility in comparison to non-fused in E. coli. The SUMO fusion also simplified the purification process. Previously engineered rabies glycoprotein gene in tobacco plants provides complete protection to mice, but the expression was very low for purification. Our finding demonstrated that the SUMO-fusion was useful for enhancing expression and solubility of the membrane protein and again proves to be a good alternative technology for applications in biomedical and pharmaceutical research.     

Revolutionizing membrane protein overexpression in bacteria

The bacterium Escherichia coli is the most widely used expression host for overexpression trials of membrane proteins. Usually, different strains, culture conditions and expression regimes are screened for to identify the optimal overexpression strategy. However, yields are often not satisfactory, especially for eukaryotic membrane proteins. This has initiated a revolution of membrane protein overexpression in bacteria. Recent studies have shown that it is feasible to (i) engineer or select for E. coli strains with strongly improved membrane protein overexpression characteristics, (ii) use bacteria other than E. coli for the expression of membrane proteins, (iii) engineer or select for membrane protein variants that retain functionality but express better than the wild-type protein, and (iv) express membrane proteins using E. coli-based cell-free systems.     

Purification of Chinese Sacbrood Virus (CSBV), Gene Cloning and Prokaryotic Expression of its Structural Protein VP1

The aim of this study was to purify the Chinese Sacbrood Virus Beijing Miyun (BJMY-CSBV) from infected Apis cerana larvae, clone structural protein gene VP1 (named BJMY-CSBV-VP1), and investigate its biological information. The result indicated that the capsid of CSBV is of spherical shape. Gene clone experiment showed that the BJMY-CSBV-VP1 gene sequence comprised 945 bp, encoding 315 amino acids with relative molecular weight of 35.59 kDa and isoelectric point 9.38 pI. Phylogenetic analysis of amino acid sequences showed that the BJMY-CSBV-VP1 and LNDD_2015 were grouped together. Protein secondary structure prediction showed that the gene contained two α-helices, thirteen β-folds, six polypeptide binding sites, and no disulfide bridge. Simultaneously, the BJMY-CSBV-VP1 was ligated to the expression vector pET32a(+) and then transformed into the Escherichia coli BL21 (DE3) for prokaryotic expression. The optimal expression experiment revealed that the protein was found in the inclusion body. The recombinant protein was successfully purified by washing buffer combined with supersonic fragmentation. In this study, we obtained the purified BJMY-CSBV particles, cloned BJMY-CSBV-VP1 gene, investigated the detailed information of the gene by analyzing the sequence, and obtained the purified recombinant protein, which could help for further understanding of the function of the structural protein gene VP1.