Cloning and expression of multiple integral membrane proteins from Mycobacterium tuberculosis in Escherichia coli

Seventy integral membrane proteins from the Mycobacterium tuberculosis genome have been cloned and expressed in Escherichia coli. A combination of T7 promoter-based vectors with hexa-His affinity tags and BL21 E. coli strains with additional tRNA genes to supplement sparsely used E. coli codons have been most successful. The expressed proteins have a wide range of molecular weights and number of transmembrane helices. Expression of these proteins has been observed in the membrane and insoluble fraction of E. coli cell lysates and, in some cases, in the soluble fraction. The highest expression levels in the membrane fraction were restricted to a narrow range of molecular weights and relatively few transmembrane helices. In contrast, overexpression in insoluble aggregates was distributed over a broad range of molecular weights and number of transmembrane helices.     

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.     

Clostridium difficile Hfq can replace Escherichia coli Hfq for most of its function

A gene for the Hfq protein is present in the majority of sequenced bacterial genomes. Its characteristic hexameric ring-like core structure is formed by the highly conserved N-terminal regions. In contrast, the C-terminal forms an extension, which varies in length, lacks homology, and is predicted to be unstructured. In Gram-negative bacteria, Hfq facilitates the pairing of sRNAs with their mRNA target and thus affects gene expression, either positively or negatively, and modulates sRNA degradation. In Gram-positive bacteria, its role is still poorly characterized. Numerous sRNAs have been detected in many Gram-positive bacteria, but it is not yet known whether these sRNAs act in association with Hfq. Compared with all other Hfqs, the C. difficile Hfq exhibits an unusual C-terminal sequence with 75% asparagine and glutamine residues, while the N-terminal core part is more conserved. To gain insight into the functionality of the C. difficile Hfq (Cd-Hfq) protein in processes regulated by sRNAs, we have tested the ability of Cd-Hfq to fulfill the functions of the E. coli Hfq (Ec-Hfq) by examining various functions associated with Hfq in both positive and negative controls of gene expression. We found that Cd-Hfq substitutes for most but not all of the tested functions of the Ec-Hfq protein. We also investigated the role of the C-terminal part of the Hfq proteins. We found that the C-terminal part of both Ec-Hfq and Cd-Hfq is not essential but contributes to some functions of both the E. coli and C. difficile chaperons.     

大肠杆菌细胞周质底物结合蛋白gsiB基因的克隆及其表达条件的优化

为深入研究大肠杆菌谷胱甘肽转运系统的蛋白质结构和功能, 对该系统中的gsiB基因进行了克隆和表达条件的优化。根据大肠杆菌谷胱甘肽转运系统中底物结合蛋白gsiB基因序列, 利用PCR方法扩增到该基因的编码区序列, 利用SLIC (Sequence and ligation–independent cloning)方法直接将其插入pWaldo-GFPe中, 成功构建了重组表达质粒pWaldo-GFP-GsiB。将重组质粒转化不同的大肠杆菌表达菌株进行诱导表达, 通过改变培养温度和IPTG浓度等条件, 得到了能够大量表达目标蛋白的重组子。结果表明: 大肠杆菌BL21(DE3)是 gsiB基因表达的最佳宿主菌; 18℃低温诱导培养有利于gsiB基因的大量表达; 0.1 mmol/L IPTG足够诱导gsiB基因表达, 增加IPTG浓度(0.1 mmol/L~1.0 mmol/L)并不能明显地促进gsiB基因的表达。Western blotting结果显示目标蛋白质有表达, 其分子量大小与预期相符。     

High-level expression and purification of Escherichia coli oligopeptidase B

Oligopeptidase B (OpdB) of Escherichia coli, previously called protease II, has a trypsin-like specificity, cleaving peptides at lysine and arginine residues and belongs to the prolyl oligopeptidase family of new serine peptidases. In this study, we report the fusion expression of E. coli oligopeptidase B with an N-terminal histidine tag using pET28a as the expression vector. Although most of the recombinant OpdB was produced as inclusion bodies, the solubility of the recombinant protease increased significantly when the expression temperature shifted from 37 to 30 degrees C. Recombinant OpdB (approximately 10 mg) could be purified from the soluble fraction of the crude extract of 1L log-phase E. coli culture containing 1.5 g wet bacterial cells. The purified OpdB has a molecular weight of approximately 80 kDa and a specific activity of 4.8 x 10(4) U/mg. OpdB could also be purified from the inclusion bodies with a lower yield. The recombinant enzyme was very stable under 40 degrees C. By comparison of the substrate specificity of the purified OpdB with that of OpdA, another trypsin-like protease in E. coli, we found that Boc-Glu-Lys-Lys-MCA is a specific substrate for E. coli OpdB. We also found that compared to OpdA, OpdB is much more sensitive to GMCHA-OPh(t)Bu, a synthetic trypsin inhibitor that can retard the growth of E. coli.     

Heterologous pyc gene expression under various natural and engineered promoters in Escherichia coli for improved succinate production

In this study, the expression level of the pyc gene from Lactococcus lactis was fine tuned to improve succinate production in Escherichia coli SBS550MG. IPTG induction in the cultures of SBS550MG with pHL413, a positive control plasmid previously constructed (Sanchez et al., 2005), gave drastically decreased PYC activity and succinate yield. We constructed several plasmids for the expression of pyc to change copy number and variant promoters. Among the constructs, as compared to pHL413, the PYC activity dropped significantly with the Plac, Ptac, Ptrc or native Ppyc promoters in medium or high copy vectors, which resulted in a decrease in succinate yield. Three constructs pThio12, pHL413-Km, and pHL413-Km(lacIq-)N showed considerable PYC activity and improved succinate production in E. coli SBS550MG. The native Ppyc promoter was also modified in order to vary pyc expression levels by site-directed mutagenesis of the −10, −35, −44 regions, and the spacer regions between −10 to −35 and −35 to −44 regions. Out of 9 native promoter variants, the MIII variant resulted in a 20% increase in PYC activity, and improved succinate yield in SBS550MG. We also determined the copy number and stability of pHL413 and pHL413-Km. The two plasmids showed roughly the same copy number, but the pHL413-Km plasmid was relatively more stable. This study provides more understanding of the plasmid characteristics and fine tuning of the expression level of pyc for optimization of the succinate production processes.     

Improved glutathione production by gene expression in Escherichia coli

AIMS: To improve glutathione (GSH) production in Escherichia coli by different genetic constructions containing GSH genes. METHODS AND RESULTS: GSH production was very low in E. coli by the expression of gshI gene. An increase of GSH production was achieved by the expression of both gshI and gshII genes in E. coli. A higher GSH production, namely 34.8 mg g(-1) wet cell weight, was obtained by simultaneous expression of two copies of gshI gene and one copy of gshII gene. CONCLUSIONS: The simultaneous expression of two copies of gshI gene and one copy of gshII gene resulted in a significant increase in GSH production. SIGNIFICANCE AND IMPACT OF THE STUDY: The expression strategy for GSH production described here can be used to increase gene expression and obtain high production rates in other multienzyme reaction systems.     

Thermostability of multidomain proteins: elongation factors EF-Tu from Escherichia coli and Bacillus stearothermophilus and their chimeric forms

Recombinant mesophilic Escherichia coli (Ec) and thermophilic Bacillus stearothermophilus (Bst) elongation factors EF-Tus, their isolated G-domains, and six chimeric EF-Tus composed of domains of either EF-Tu were prepared, and their GDP/GTP binding activities and thermostability were characterized. BstEF-Tu and BstG-domain bound GDP and GTP with affinities in nanomolar and submicromolar ranges, respectively, fully comparable with those of EcEF-Tu. In contrast, the EcG-domain bound the nucleotides with much lower, micromolar affinities. The exchange of domains 2 and 3 had essentially no effect on the GDP-binding activity; all complexes of chimeric EF-Tus with GDP retained K(d) values in the nanomolar range. The final thermostability level of either EF-Tu was the result of a cooperative interaction between the G-domains and domains 2 + 3. The G-domains set up a "basic" level of the thermostability, which was approximately 20 degrees C higher with the BstG-domain than with the EcG-domain. This correlated with the growth temperature optimum difference of both bacteria and two distinct thermostabilization features of the BstG-domain: an increase of charged residues at the expense of polar uncharged residues (CvP bias), and a decrease in the nonpolar solvent-accessible surface area. Domains 2 + 3 contributed by further stabilization of alpha-helical regions and, in turn, the functions of the G-domains to the level of the respective growth temperature optima. Their contributions were similar irrespective of their origin but, with Ecdomains 2 + 3, dependent on the guanine nucleotide binding state. It was lower in the GTP conformation, and the mechanism involved the destabilization of the alpha-helical regions of the G-domain by Ecdomain 2.     

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has become the method of choice for introducing DNA into prokaryotes that are not naturally competent. Electroporation is a rapid, efficient, and streamlined transformation method that, in addition to purified DNA and competent bacteria, requires commercially available gene pulse controller and cuvettes. In contrast to the pulsing step, preparation of electrocompetent cells is time consuming and labor intensive involving repeated rounds of centrifugation and washes in decreasing volumes of sterile, cold water, or non-ionic buffers of large volumes of cultures grown to mid-logarithmic phase of growth. Time and effort can be saved by purchasing electrocompetent cells from commercial sources, but the selection is limited to commonly employed E. coli laboratory strains. We are hereby disseminating a rapid and efficient method for preparing electrocompetent E. coli, which has been in use by bacteriology laboratories for some time, can be adapted to V. cholerae and other prokaryotes. While we cannot ascertain whom to credit for developing the original technique, we are hereby making it available to the scientific community.     

Structure of the cyclomodulin Cif from pathogenic Escherichia coli

Bacterial pathogens have evolved a sophisticated arsenal of virulence factors to modulate host cell biology. Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) use a type III protein secretion system (T3SS) to inject microbial proteins into host cells. The T3SS effector cycle inhibiting factor (Cif) produced by EPEC and EHEC is able to block host eukaryotic cell-cycle progression. We present here a crystal structure of Cif, revealing it to be a divergent member of the superfamily of enzymes including cysteine proteases and acetyltransferases that share a common catalytic triad. Mutation of these conserved active site residues abolishes the ability of Cif to block cell-cycle progression. Finally, we demonstrate that irreversible cysteine protease inhibitors do not abolish the Cif cytopathic effect, suggesting that another enzymatic activity may underlie the biological activity of this virulence factor.     

Molecular Characterization of an Ice Nucleation Protein Variant (InaQ) from Pseudomonas syringae and the Analysis of Its Transmembrane Transport Activity in Escherichia coli

The ice nucleation protein (INP) of Pseudomonas syringae has gained scientific interest not only because of its pathogenicity of foliar necroses but also for its wide range of potential applications, such as in snow making, frozen food preparation, and surface-display system development. However, studies on the transport activity of INP remain lacking. In the present study, a newly identified INP-gene variant, inaQ, from a P. syringae MB03 strain was cloned. Its structural domains, signal sequences, and the hydrophilicity or hydrophobicity of each domain, were then characterized. The deduced amino acid sequence of InaQ shares similar protein domains with three P. syringae INPs, namely, InaK, InaZ, and InaV, which were identified as an N-terminal domain, a central repeating domain, and a C-terminal domain. The expression of the full-length InaQ and of various truncated variants was induced in Escherichia coli to analyze their transmembrane transport and surface-binding activities, while using the green fluorescence protein (GFP) as the fusion partner. With two transmembrane segments and a weak secretion signal, the N-terminal domain (InaQ-N) alone was found to be responsible for the transport process as well as for the binding to the outer membrane, whereas the C-terminal region was nonfunctional in protein transport. Increased membrane transport and surface-binding capacities were induced by a low isopropyl-β-D-thiogalactoside concentration (0.1 mmol/l) but not by culture temperatures (15 ºC to 37 ºC). Furthermore, by constructing the GFP-fused proteins with a single InaQ-N, as well as two and three tandemly aligned InaQ-N molecules, the transport and membrane-binding activities of these proteins were compared using Western blot analysis, immmunofluorescence microscopy, and assays of the GFP specific fluorescence intensity of subcellular fractions and flow cytometry, which showed that the increase of InaQ-N repeats resulted in a coordinated increase of the surface-immobilization efficiency. Therefore, the results of this study can serve as a molecular basis for improving the performance of INP-based cell surface-display systems.     

Interaction of Escherichia coli K1 and K5 with Acanthamoeba castellanii trophozoites and cysts

The existence of symbiotic relationships between Acanthamoeba and a variety of bacteria is well-documented. However, the ability of Acanthamoeba interacting with host bacterial pathogens has gained particular attention. Here, to understand the interactions of Escherichia coli K1 and E. coli K5 strains with Acanthamoeba castellanii trophozoites and cysts, association assay, invasion assay, survival assay, and the measurement of bacterial numbers from cysts were performed, and nonpathogenic E. coli K12 was also applied. The association ratio of E. coli K1 with A. castellanii was 4.3 cfu per amoeba for 1 hr but E. coli K5 with A. castellanii was 1 cfu per amoeba for 1 hr. By invasion and survival assays, E. coli K5 was recovered less than E. coli K1 but still alive inside A. castellanii. E. coli K1 and K5 survived and multiplied intracellularly in A. castellanii. The survival assay was performed under a favourable condition for 22 hr and 43 hr with the encystment of A. castellanii. Under the favourable condition for the transformation of trophozoites into cysts, E. coli K5 multiplied significantly. Moreover, the pathogenic potential of E. coli K1 from A. castellanii cysts exhibited no changes as compared with E. coli K1 from A. castellanii trophozoites. E. coli K5 was multiplied in A. castellanii trophozoites and survived in A. castellanii cysts. Therefore, this study suggests that E. coli K5 can use A. castellanii as a reservoir host or a vector for the bacterial transmission.     

Increased protein yields from Escherichia coli using pressure-cycling technology.

Sample preparation is critical to the success of two-dimensional gel electrophoresis and other analytical methods. Pressure-cycling technology (PCT) uses alternating cycles of high and low pressure to induce cell lysis. Cell suspensions were placed in PULSE Tubes and subjected to alternating cycles of high and low pressure in a Barocycler instrument. each cycle consisted of 20 sec at 35,000 psi followed by 20 sec at ambient pressure. For the bacterium Escherichia coli, PCT extracted 14.2% more total protein than was extracted using a standard bead mill. Image analysis of two-dimensional gels revealed 801 protein spots in the PCT lysate, compared to 760 protein spots in the bead mill lysate.     

Intracellular monitoring of superoxide dismutase expression in an Escherichia coli fed-batch cultivation using on-line disruption with at-line surface plasmon resonance detection

An on-line cell disruption system for at-line monitoring of the intracellular concentration of recombinant human superoxide dismutase (rhSOD) in a genetically modified Escherichia coli strain, HMS174(DE3) (pET11a/rhSOD), in bioreactor cultivations is described. The sampled bacteria were disrupted on-line by rapid mixing with a nonionic detergent. The recombinant protein content of the lysed bacterial sample was quantitated by a subsequent surface plasmon resonance biosensor with a specific monoclonal antibody. Extraction efficiency of the monitoring system was optimized with respect to the flow rate ratio of the cell suspension and the detergent at relevant cell densities with the aim to attain rapid monitoring. Monitoring was demonstrated for a shake flask culture and a glucose-limited fed-batch cultivation. The results are compared with a traditional enzyme-linked immunosorbent assay method showing a correlation coefficient of R2 = 0.97. Extraction efficiency of rhSOD reached 95-99% at a total processing time of 1.8-2.6 min and a contact time of 0.8-1.4 min. The possibility of extending the monitoring system to other intracellular proteins is discussed.     

Quantitative export of a reporter protein,GFP, by the twin-arginine translocation pathway in Escherichia coli

The Tat system mediates the transport of folded proteins across the bacterial cytoplasmic membrane. To study the properties of the Escherichia coli Tat-system, we used green fluorescent protein (GFP) fused to the twin-arginine signal peptide of TMAO reductase (TorA). In the presence of arabinose, low levels of this protein rapidly saturate the translocase and cause the accumulation of inactive, membrane-bound TorA-GFP; fluorescence microscopy also showed active TorA-GFP to be distributed throughout the cytoplasm. However, the efficiency of export can be massively increased by alteration of the growth conditions, and further increased by overexpression of the tatABC genes. Under these conditions, the levels of GFP in the periplasm are raised over 20-fold and the export efficiency nears 100%. These results show that the Tat-system is relatively inactive under some growth conditions and the data suggest that the system may be applicable for the larger-scale export of heterologous proteins.