Expression of Semliki Forest virus E1 protein in Escherichia coli. Low pH-induced pore formation

Exposure of Semliki Forest virus 1 to mildly acidic conditions results in conformational changes of the viral spike proteins, which in turn leads to a pore formation across its membrane. The ability to form a pore has been ascribed to the ectodomain of the Semliki Forest virus (SFV) E1 spike protein. To elucidate whether the E1 protein per se is sufficient for low pH-dependent pore formation, we expressed E1 in Escherichia coli in an inducible manner using the pET11c expression system. The data obtained clearly showed that the E1 protein was expressed in the bacterial cell membrane and that exposure of E. coli expressing the SFV E1 protein to low pH (<6.2) resulted in a permeability change of the membrane. Thus, we conclude that the E1 protein of SFV per se is sufficient to promote pore formation under mildly acidic conditions.     

The spike protein of severe acute respiratory syndrome (SARS) is cleaved in virus infected Vero-E6 cells

INTRODUCTION The severe acute respiratory syndrome (SARS), causedby SARS-associated coronavirus (SARS-CoV) [1], wasrecently identified as a new clinical entity. It apparentlybegan in Guangdong province of China in November of2002 and has spread to sever…     

Truncated hemoglobin o of Mycobacterium tuberculosis: the oligomeric state change and the interaction with membrane components

Being an obligate aerobe, the Mycobacterium tuberculosis cells would have to evolve a mechanism to collect and deliver the hardly available O(2) to survive in granulomas and to maintain the low level of respiration during latency. The M. tuberculosis truncated hemoglobin o (trHbO), when heterologously expressed in Escherichia coli cells, was found to significantly enhance the cellular respiration and cell growth. This study was undertaken in an attempt to understand the molecular details for trHbO to promote the cellular respiration, focusing on the ways through which trHbO is recruited to the cell membrane and O(2) molecules are delivered. Our data demonstrate that the trHbO protein is able to promote the growth of E. coli cells in a fashion that depends on the presence of the respiratory chain terminal oxidase cytochrome o complex (or Cyo complex). The trHbO protein appears to interact with the Cyo B subunit of the Cyo complex directly, likely in a dynamic manner. The trHbO is also able to bind membrane lipids in a non-specific way, during the process electrostatic and hydrophobic interactions both likely exist. Besides, binding with membrane induces the dissociation of trHbO from dimers to monomers. In light of these observations, a hypothesis was made to explain how trHbO might serve as an O(2) collector and/or reservoir for M. tuberculosis cells under O(2)-limiting or lacking conditions.     

SidK-VatA蛋白复合物的表达和纯化

军团菌以吞噬泡的形式进入宿主细胞,借助宿主细胞环境进行自身的繁殖传代。为避免被宿主识别,通过分泌自身效应蛋白来逃避宿主对含病原菌液泡（legionella containing vacuoles,LCVs）的识别和消化,军团菌通过分泌效应蛋白SidK结合宿主v-ATPase,抑制v-ATPase对吞噬泡的酸化,以实现吞噬泡不被溶酶体消化。在大肠杆菌中表达了效应蛋白SidK,利用昆虫细胞表达了VatA（v-ATPase）蛋白,得到了均一性较好的SidK-VatA蛋白复合体,为进一步解析这个复合体的结构奠定了基础。     

A novel system for expressing recombinant proteins over a wide temperature range from 4 to 35 degrees C

Escherichia coli cells are the most commonly used host cells for large-scale production of recombinant proteins, but some proteins are difficult to express in E. coli. Therefore, we tested the nocardioform actinomycete Rhodococcus erythropolis, which grows at temperatures ranging from 4 to 35 degrees C, as an expression host cell. We constructed inducible expression vectors, where the expression of the target genes could be controlled with the antibiotic thiostrepton. Using these expression vectors, several milligrams of reporter proteins could be isolated from 1 liter of culture of R. erythropolis cells grown at a temperature range from 4 to 35 degrees C. Moreover, we successfully purified serum amyloid A1, NADH dehydorogenase 1 alpha subcomplex 4, cytochrome b5-like protein, apolipoprotein A-V, cathepsin D, pancreatic Rnase, and HMG-1 that are all difficult to express in E. coli. In the case of kallikrein 6, mouse deoxyribonuclease I and Kid1, which are also difficult to express in E. coli, the expression level of each protein increased when proteins were expressed at low temperature (4 degrees C). Based on these results, we conclude that a recombinant protein expression system using R. erythropolis as the host cell is superior to respective E. coli systems.     

Viroporin activity of murine hepatitis virus E protein

The viroporin activity of the E protein from murine hepatitis virus (MHV), a member of the coronaviruses, was analyzed. Viroporins are a growing family of viral proteins able to enhance membrane permeability, promoting virus budding. Initially, the MHV E gene was inducibly expressed in Escherichia coli cells, leading to the arrest of bacterial growth, cell lysis and permeabilization to different compounds. Thus, exit of labeled nucleotides from E. coli cells to the cytoplasm was apparent upon expression of MHV E. In addition, enhanced entry of the antibiotic hygromycin B occurred at levels comparable to those observed with the viroporin 6K from Sindbis virus. Mammalian cells are also readily permeabilized by the expression of MHV E protein. Finally, brefeldin A powerfully blocks the viroporin activity of the E protein in BHK cells, suggesting that an intact vesicular system is necessary for this coronavirus to permeabilize mammalian cells.     

E. coli filament formation induced by heterologous S-layer expression

Escherichia coli is a rod-shaped intestinal bacterium which has a size of 1.1-1.5 μm x 2.0-6.0?μm. The fast cell division process and the uncomplicated living conditions have turned E. coli into a widely used host in genetic engineering and into one of the best studied microorganisms of all. We used E. coli BL21(DE3) as host for heterologous expression of S-layer proteins of Lysinibacillus sphaericus JG-A12 in order to enable a fast and high efficient protein production. The S-layer expression induced in E. coli an unusual elongation of the cells, thus producing filaments of > 100 μm in length. In the stationary growth phase, E. coli filaments develop tube-like structures that contain E. coli single cells. Fluorescence microscopic analyses of S-layer expressing E. coli cells that were stained with membrane stain FM (?) 5-95 verify the membrane origin of the tubes. Analyses of DAPI stained GFP-S-layer expressing E. coli support the assumption of a disordered cell division that is induced by the huge amount of recombinant S-layer proteins. However, the underlying mechanism is still not characterized in detail. These results describe the occurrence of a novel stable cell form of E. coli as a result of a disordered cell division process.     

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.     

Membrane permeabilization by different regions of the human immunodeficiency virus type 1 transmembrane glycoprotein gp41.

The transmembrane glycoprotein (gp41) of human immunodeficiency virus type 1 (HIV-1) has been implicated in the cytopathology observed during HIV infection. The first amino acids located at the amino terminus are involved in membrane fusion and syncytium formation, while sequences located at the carboxy terminus have been predicted to interact with membranes and modify membrane permeability. The HIV-1 gp41 gene has been cloned and expressed in Escherichia coli cells by using pET vectors to analyze changes in membrane permeability produced by this protein. This system is well suited for expressing toxic genes in an inducible manner and for analyzing the function of proteins that modify membrane permeability. gp41 enhances the permeability of the bacterial membrane to hygromycin B despite the low level of expression of this protein. To localize the regions of gp41 responsible for these effects, a number of fragments spanning different portions of gp41 were inducibly expressed in E. coli. Two regions of gp41 were shown to increase membrane permeability: one located at the carboxy terminus, where two highly amphipathic helices have been predicted, and another one corresponding to the membrane-spanning domain. Expression of the central region of gp41 comprising this domain was highly lytic for E. coli cells and increased membrane permeability to a number of compounds. These findings are discussed in the light of HIV-induced cytopathology and gp41 structure.     

利用双启动子表达载体pDH2-YggG-Ptac-Era研究E. coli Era和YggG 蛋白间的相互关系

yggG是从大肠杆菌全基因组文库中钓取并克隆的Era结合蛋白基因,研究表明该基因表达的YggG294(amino acids 1-294)蛋白对宿主菌的生长具有强烈的抑制作用。为了阐明YggG与Era间的相互关系,构建可同时可控性表达Era和YggG294蛋白的双启动子表达载体。利用所构建的双启动子表达载体在同一细胞中同时可控性地表达YggG294与Era蛋白。结果显示,在不表达和少量表达YggG294的细菌细胞内,Era 的表达量与总蛋白量的比值随着诱导时间增加而增高,而YggG294大量表达的细菌内Era 的表达量与总蛋白量的比值基本保持不变;Era 蛋白的预表达对YggG294表达所引起的细菌生长率下降无影响。由此可以推论,YggG294的过表达引起宿主菌生长抑制进而影响了Era蛋白的进一步表达,而YggG294的过表达引起宿主菌生长抑制与YggG和Era蛋白间的相互作用无关     

四种类型启动子控制下空肠弯曲杆菌血红蛋白对大肠杆菌生长速度的影响

细菌血红蛋白可增加细胞在低氧条件下的氧气利用率,提高细胞的增长速率。为提高E. coli在现有发酵水平下的生物量,获得新型的基因工程底盘细胞,基于空肠弯曲杆菌(Campylobacter jejuni)血红蛋白基因,比较分析了组成型和诱导型启动子对血红蛋白在E. coli基因工程菌中的表达状况及对E. coli生长的影响。首先合成了空肠弯曲杆菌血红蛋白基因chb,并让chb基因在诱导型启动子(P_T7和P_vgh)和组成型启动子(P2和P_spoI-Ⅱ)控制下表达;随后,测定了由四种类型启动子控制的血红蛋白工程菌在摇瓶和蓝盖瓶中的生长状况。结果表明四种重组菌可以表达出有活性的血红蛋白,并可显著提高大肠杆菌的生长;在发酵罐中的试验也表明四种工程菌同样对细胞生长具有出促进作用;进一步比较分析了菌体密度、细胞鲜重和CO差式光谱值间的相互关系,讨论了四种类型启动子控制的基因工程菌在诱导表达和生长调控等方面的特点,并为在微氧和好氧等不同发酵条件下选用基因工程底盘细胞提供了参考。     

Heterologous expression and localization of gentisate transporter Ncg12922 from Corynebacterium glutamicum ATCC 13032

Ralstonia sp. strain U2 metabolizes naphthalene via gentisate (2,5-dihydroxybenzoate) to central metabolites, but it was found unable to utilize gentisate as growth substrate. A putative gentisate transporter encoded by ncg12922 from Corynebacterium glutamicum ATCC 13032 was functionally expressed in Ralstonia sp. strain U2, converting strain U2 to a gentisate utilizer. After ncg12922 was inserted into plasmid pGFPe with green fluorescence protein gene gfp, the expressed fusion protein Ncg12922-GFP could be visualized in the periphery of Escherichia coli cells under confocal microscope, consistent with a cytoplasmic membrane location. In contrast, GFP was ubiquitous in the cytoplasm of E. coli cells carrying pGFPe only. Gentisate 1,2-dioxygenase activity was present in the cell extract from strain U2 induced with gentisate but at a much lower level (one-fifth) than that obtained with salicylate. However, it exhibited a similar level in strain U2 containing Ncg12922 induced either by salicylate or gentisate.     

Expression of recombinant EARLI1, a hybrid proline-rich protein of Arabidopsis, in Escherichia coli and its inhibition effect to the growth of fungal pathogens and Saccharomyces cerevisiae

EARLI1 is an Arabidopsis gene with pleiotropic effects previously shown to have auxiliary functions in protecting plants against freezing-induced cellular damage and promoting germinability under low-temperature and salinity stresses. Here we determined whether recombinant EARLI1 protein has anti-fungal activity. Recombinant EARLI1 protein lacking its signal peptide was produced in Escherichia coli BL21(DE3) using isopropyl β-d-1-thiogalactopyranoside (IPTG) induction and the prokaryotic expression vector pET28a. Expression of EARLI1 was analyzed by Western blotting and the protein was purified using affinity chromatography. Recombinant EARLI1 protein was applied to fungal cultures of Saccharomyces cerevisiae, Botrytis cinerea and Fusarium oxysporum, and membrane permeability was determined using SYTOX green. Full-length EARLI1 was expressed in S. cerevisiae from the GAL1 promoter using 2% galactose and yeast cell viability was compared to control cells. Our results indicated that application of recombinant EARLI1 protein to B. cinerea and F. oxysporum could inhibit the growth of the necrotrophic fungi. Besides, addition of the recombinant protein to liquid cultures of S. cerevisiae significantly suppressed yeast growth and cell viability by increasing membrane permeability, and in vivo expression of the secreted form of EARLI1 in S. cerevisiae also had a remarkable inhibition effect on the growth of yeast cells.     

微波杀菌过程中大肠杆菌与金黄色葡萄球菌细胞膜通透性的改变

对细胞膜通透性变化的研究是认识微波杀菌机理的途径之一。用荧光探针检测微波处理后细胞内Ca2 浓度的变化,可以精确地表征细胞膜通透性的改变。选用二乙酸荧光素(FDA)和Fluo-3/AM两种荧光染料,对大肠杆菌(Escherichiacoli)和金黄色葡萄球菌(Staphylococcus aureus)经微波处理后的酯酶活性及细胞膜通透性进行研究,结果表明大肠杆菌与金黄色葡萄球菌的胞内非特异性酯酶(NSE)活性及细胞膜通透性的变化情形有所不同。在50℃、55℃、60℃和65℃微波处理条件下,大肠杆菌细胞膜通透性分别增加了20.7%、28.1%、74.8%、89.8%,而金黄色葡萄球的增加不显著,分别比对照组提高了4.1%、6.0%、21.9%和19.7%。细胞膜通透性的改变与微生物致死率有一定的相关性,也可能是微波杀菌非热效应的表现之一。     

Incorporation of heterologous outer membrane and periplasmic proteins into Escherichia coli outer membrane vesicles

Gram-negative bacteria shed outer membrane vesicles composed of outer membrane and periplasmic components. Since vesicles from pathogenic bacteria contain virulence factors and have been shown to interact with eukaryotic cells, it has been proposed that vesicles behave as delivery vehicles. We wanted to determine whether heterologously expressed proteins would be incorporated into the membrane and lumen of vesicles and whether these altered vesicles would associate with host cells. Ail, an outer membrane adhesin/invasin from Yersinia enterocolitica, was detected in purified outer membrane and in vesicles from Escherichia coli strains DH5alpha, HB101, and MC4100 transformed with plasmid-encoded Ail. In vesicle-host cell co-incubation assays we found that vesicles containing Ail were internalized by eukaryotic cells, unlike vesicles without Ail. To determine whether lumenal vesicle contents could be modified and delivered to host cells, we used periplasmically expressed green fluorescent protein (GFP). GFP fused with the Tat signal sequence was secreted into the periplasm via the twin arginine transporter (Tat) in both the laboratory E. coli strain DH5alpha and the pathogenic enterotoxigenic E. coli ATCC strain 43886. Pronase-resistant fluorescence was detectable in vesicles from Tat-GFP-transformed strains, demonstrating that GFP was inside intact vesicles. Inclusion of GFP cargo increased vesicle density but did not result in morphological changes in vesicles. These studies are the first to demonstrate the incorporation of heterologously expressed outer membrane and periplasmic proteins into bacterial vesicles.     

Chlorination and ozonation of waste-water: comparative analysis of efficacy through the effect on Escherichia coli membranes

The effect of chlorine and ozone on Escherichia coli cells resuspended in waste-water was compared. Selected chlorination and ozonation conditions produced a similar decrease in culturability (2-2.5 log). Under these conditions, differences in membrane permeability and cell surface hydrophobicity, depending on the disinfectant tested, were detected. After ozonation, while no changes in cell surface hydrophobicity were observed, approximately 95.5% of cells showed altered membrane permeability. The effect of chlorine was not linked to changes in membrane permeability. After chlorination, E. coli cells showed a tendancy to aggregate. The possibility that aggregation of cells could interfere with conventional colony counts is discussed. The degree of toxicity (Microtox assay) was unrelated to the effect on cellular activity.     

重组人钙网蛋白的克隆与原核表达

［摘要］目的: 克隆人钙网蛋白（calreticulin,CRT）并在E.coli中原核表达和纯化。方法：采用RT-PCR 法从人非小细胞肺腺癌A549细胞总RNA中克隆人钙网蛋白cDNA,构建CRT原核表达质粒（pET-15b/CRT）并转化E.coli 的Rossetta菌株。IPTG诱导后,表达蛋白在变性条件下经Ni-NTA 树脂亲和层析纯化,然后透析复性。分别用SDS-PAGE和Western blotting法鉴定CRT表达和纯化状态。结果：从A549细胞总RNA中成功获得人CRT cDNA克隆,重组质粒pET-15b/CRT构建正确。转化pET-15b/CRT的E.coli Rossetta诱导性表达重组人CRT蛋白,该蛋白可经Ni-NTA树脂亲和层析高度纯化。结论：成功建立了CRT原核表达和纯化的实验方法,该方法为后续的CRT蛋白功能研究奠定了基础。     

Comparative analysis and "expression space" coverage of the production of prokaryotic membrane proteins for structural genomics

Membrane proteins comprise up to one-third of prokaryotic and eukaryotic genomes, but only a very small number of membrane protein structures are known. Membrane proteins are challenging targets for structural biology, primarily due to the difficulty in producing and purifying milligram quantities of these proteins. We are evaluating different methods to produce and purify large numbers of prokaryotic membrane proteins for subsequent structural and functional analysis. Here, we present the comparative expression data for 37 target proteins, all of them secondary transporters, from the mesophilic organism Salmonella typhimurium and the two hyperthermophilic organisms Aquifex aeolicus and Pyrococcus furiosus in three different Escherichia coli expression vectors. In addition, we study the use of Lactococcus lactis as a host for integral membrane protein expression. Overall, 78% of the targets were successfully produced under at least one set of conditions. Analysis of these results allows us to assess the role of different variables in increasing "expression space" coverage for our set of targets. This analysis implies that to maximize the number of nonhomologous targets that are expressed, orthologous targets should be chosen and tested in two vectors with different types of promoters, using C-terminal tags. In addition, E. coli is shown to be a robust host for the expression of prokaryotic transporters, and is superior to L. lactis. These results therefore suggest appropriate strategies for high-throughput heterologous overproduction of membrane proteins.     

Construction of phoE-caa, a novel PCR- and immunologically detectable marker gene for Pseudomonas putida.

In this paper we describe the construction and use in Pseudomonas putida WCS358 of phoE-caa, a novel hybrid marker gene, which allows monitoring both at the protein level by immunological methods and at the DNA level by PCR. The marker is based on the Escherichia coli outer membrane protein gene phoE and 75 bp of E. coli caa, which encode a nonbacteriocinic fragment of colicin A. This fragment contains an epitope which is recognized by monoclonal antibody (MAb) 1C11. As the epitope is contained in one of the cell surface-exposed loops of PhoE, whole cells of bacteria expressing the protein can be detected by using the MAb. The marker gene contains only E. coli sequences not coding for toxins and therefore can be considered environmentally safe. The hybrid PhoE-ColA protein was expressed in E. coli under conditions of phosphate starvation, and single cells could be detected by immunofluorescence microscopy with MAb 1C11. Using a wide-host-range vector the phoE-caa gene was introduced into P. putida WCS358. The gene appeared to be expressed under phosphate limitation in this species, and the gene product was present in the membrane fraction and reacted with MAb 1C11. The hybrid PhoE-ColA protein could be detected on whole cells of WCS358 mutant strains lacking (part of) the O-antigen of the lipopolysaccharide but not on wild-type WCS358 cells, unless these cells had previously been washed with 10 mM EDTA. In addition to immunodetection, the phoE-caa marker gene could be specifically detected by PCR with one primer directed to a part of the phoE sequence and a second primer that annealed to the caa insert.     

Abnormal cell division caused by inclusion bodies in E. coli; increased resistance against external stress

Inclusion body formation occurs naturally in prokaryotic cells, but is particularly common when heterologous foreign proteins are overexpressed in bacterial systems. The plant disease virus protein CMV 3a (cucumber mosaic virus movement protein) and the 56 kDa Orientia tsutsugamushi (OT56) protein (an outer membrane protein), which causes tsutsugamushi disease, were expressed in Escherichia coli, and found to form inclusion bodies. Confocal laser scanning microscopy revealed that these inclusion bodies are localized at the cellular poles within E. coli. Cells expressing inclusion bodies appeared to be interconnected, and divided abnormally. The clustered cells exhibited biofilm-like characteristics in that the interior cells of the community were protected by the antibiotic resistance of the outer cells. We compared the number of colony-forming units in inclusion body-forming versus non-forming E. coli to demonstrate the effects of lysozyme, sonication or antibiotic treatment. E. coli clustering provided significantly improved protection against cell disruption/lysis by physical and biochemical stress. This is the first report that shows that abnormal cell division caused by inclusion body formation can cause cellular clustering, resulting in improved resistance to stress in vitro.