• Journal of Marine Bioscience and Biotechnology
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• v.2 no.2
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• pp.123-125
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• 2007

Vibrio anguillarum is a gram-negative bacterium that causes vibriosis in approximately 80 different fish species. V. anguillarum produces several exotoxins are correlated with the pathogenesis of vibriosis. This study is focused on the composition of the outer membrane vesicle. Most of gram-negative bacteria produce outer membrane vesicle (OMV) during cell growth. OMV was formed from the outer membrane surface of cell and than released to extracellular environment. OMV consists of outer membrane lipids, outer membrane protein (OMP), LPS, and soluble periplasmic components. Also, they contain toxins, adhesions, and immunomodulatory. Many gram-negative bacteria were studied out forming OMV. In Vibrio sp., formation of OMV by electron microscopy has been reported from V. cholerae and V. parahaemolyticus. In present study, we isolated OMV from V. anguillarum and OMV protein was separated by SDS-PAGE. Magor band was sliced and analyzed by MALDI-TOF. The major protein band of 38kDa was identified as OmpU by MALDI-TOF MS analysis.

• Journal of Photoscience
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• v.9 no.2
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• pp.275-277
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• 2002

We have recently found that a detergent-resistant raft like membrane (DRM) can be prepared from bovine rod outer segment membranes as a low-density buoyant fraction in sucrose density gradient ultracentrifugation. G protein (transducin) and its effector enzyme (phosphodiesterase: PDE) drastically change their affinities to DRM in the process of phototransduction. We report here that the recruitment of transducin and/or $^2$PDE to DRM has close relationship with their states in signal transduction. Active T$\alpha$/PDE-complex has a high affinity to DRM, whereas inactive transducin, or inactive PDE are excluded from DRM. Active T$\alpha$/PDE-complex seems to bind to a GTPase activating protein (GRS9) in multi- protein complexes localized on DRM. Physiological significance of the multi-protein complex on the raft-like membrane in vertebrate phototransduction would be discussed.

• Journal of Microbiology
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• v.43 no.5
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• pp.437-442
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• 2005

Sera from rabbits were infected with Vibrio vulnificus containing an antibody against major outer membrane protein (MOMP). MOMP of V. vulnificus ATCC 27562 were isolated and purified by Sarkosyl and TritonX-100 dual treatment. Molecular size of MOMP was identified as 36-kDa on $13\%$ SDS-PAGE. The sequence of the first 26 amino acid residues from the N-terminal end of the protein is AELYNQDGTSLDMGGRAEARLSMKDG, which is a perfect match with OmpU of V. vulnificus CMCP6 and YJ016. MOMP specific IgM and IgG were investigated in groups of mice. The group of mice immunized with MOMP and Alum showed higher levels of IgG2b than the group immunized with only MOMP. Vaccination with MOMP resulted in protective antibodies in the mouse infection experiment.

• Journal of Microbiology and Biotechnology
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• v.29 no.1
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• pp.1-10
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• 2019

Gram-negative pathogens, such as Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii, pose a serious threat to public health worldwide, due to high rates of antibiotic resistance and the lack of development of novel antimicrobial agents targeting Gram-negative bacteria. The outer membrane (OM) of Gram-negative bacteria is a unique architecture that acts as a potent permeability barrier against toxic molecules, such as antibiotics. The OM is composed of phospholipids, lipopolysaccharide (LPS), outer membrane ${\beta}-barrel$ proteins (OMP), and lipoproteins. These components are synthesized in the cytoplasm or in the inner membrane, and are then selectively transported to the OM by the specific transport machines, including the Lol, BAM, and Lpt pathways. In this review, we summarize recent studies on the assembly systems of OM components and analyze studies for the development of inhibitors that target these systems. These analyses show that OM assembly machines have the potential to be a novel attractive drug target of Gram-negative bacteria.

• Microbiology and Biotechnology Letters
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• v.34 no.3
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• pp.273-277
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• 2006

To investigate the antigenicity and protective immunity of outer membrane protein H (OmpH) in Pasteurella multocida D:4, the recombinant OmpH protein was produced in Escherichia coli. The truncated and Trx-fused form of recombinant OmpH (53 kDa) was purified, and used as an antigen in the immunization and challenge experiment. The immunized mice with the recombinant OmpH produced a high-titer antibody, and had protective immunity against P. multocida as same level as the mice immunized with formalin-killed whole cell.

• Journal of Microbiology
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• v.45 no.2
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• pp.179-184
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• 2007

Pasteurella multocida, a Gram-negative facultative anaerobic bacterium, is a causative animal pathogen in porcine atrophic rhinitis and avian fowl cholera. For the development of recombinant subunit vaccine against P. multocida, we cloned and analyzed the gene for outer membrane protein H (ompH) from a native strain of Pasteurella multocida in Korea. The OmpH had significant similarity in both primary and secondary structure with those of other serotypes. The full-length, and three short fragments of ompH were expressed in E. coli and the recombinant OmpH proteins were purified, respectively. The recombinant OmpH proteins were antigenic and detectable with antisera produced by either immunization of commercial vaccine for respiratory disease or formalin-killed cell. Antibodies raised against the full-length OmpH provided strong protection against P. multocida, however, three short fragments of recombinant OmpHs, respectively, showed slightly lower protection in mice challenge. The recombinant OmpH might be a useful vaccine candidate antigen for P. multocida.

• Journal of Photoscience
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• v.9 no.2
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• pp.47-50
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• 2002

Raft is a distinctive membrane domain enriched in a certain class of lipids, cholesterol, and proteins observed on the plasma membrane. Growing evidence has revealed that such membrane domains play key roles in signal transduction, fertilization, development, transmitter release, and so on. Recently, we have isolated raft-like detergent-resistant membrane (DRM) fraction from bovine photoreceptor rod outer segments. Transducin and its effecter, cGMP-phosphodiesterase, elicited stimulus-dependent translocation between detergent-soluble membrane and DRM. This suggested potential importance of such distinct membrane domains in vertebrate phototransduction. Here, we will discuss physiological meaning of the translocation of major components of cGMP cascade to raft-like membrane in phototransduction. We would like to propose a hypothesis that raft-like membrane domains on the disk membrane are the place where cGMP cascade system could be quenched.

• Journal of Microbiology and Biotechnology
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• v.18 no.5
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• pp.845-851
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• 2008

TolC is an outer membrane porin protein and an essential component of drug efflux and type-I secretion systems in Gram-negative bacteria. TolC comprises a periplasmic $\alpha$-helical barrel domain and a membrane-embedded $\beta$-barrel domain. TdeA, a functional and structural homolog of TolC, is required for toxin and drug export in the pathogenic oral bacterium Actinobacillus actinomycetemcomitans. Here, we report the expression of the periplasmic domain of TdeA as a soluble protein by substitution of the membrane-embedded domain with short linkers, which enabled us to purify the protein in the absence of detergent. We confirmed the structural integrity of the TdeA periplasmic domain by size-exclusion chromatography, circular dichroism spectroscopy, and electron microscopy, which together showed that the periplasmic domain of the TolC protein family fold correctly on its own. We further demonstrated that the periplasmic domain of TdeA interacts with peptidoglycans of the bacterial cell wall, which supports the idea that completely folded TolC family proteins traverse the peptidoglycan layer to interact with inner membrane transporters.

• Korean Journal of Veterinary Research
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• v.46 no.1
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• pp.13-19
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• 2006

Generally known psittacosis or ornithosis is a disease of birds caused by the bacterium Chlamydia psittaci. Humans are accidential hosts and are most commonly infected from avian sources. It raises hepatitis or neurosis. As major outer membrane protein (MOMP) of Chlamydia psittaci has been known to play a role in the avoidance of host immune defenses, research on developing a Chlamydia vaccine has focused on the MOMP. In this study, the gene encoding the major outer membrane protein (MOMP) of the Chlamydia psittaci strain 6BC was cloned and expressed in Escherichia coli strain M-15. The recombinant DNA was cloned by fusion prokaryotic expression vector pQE30-GFPII. Expression of the recombinant protein was performed in E. coli and was induced by IPTG. The size of expressed recombinant protein is 74.220 kDa (MOMP, 43.260 kDa; GFP expression region, 30 kDa; $6{\times}His$ tag, 960Da). This protein was purified by using his-tagging-inclusion body. Recombinant protein was reconfirmed through ELISA test and western blot with antibody against pQE30-GFPII. It will be useful antibody development.

• Journal of Microbiology and Biotechnology
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• v.32 no.5
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• pp.621-629
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• 2022

Bacterial outer membrane vesicles (OMVs) typically contain multiple immunogenic molecules that include antigenic proteins, making them good candidates for vaccine development. In animal models, vaccination with OMVs has been shown to confer protective immune responses against many bacterial diseases. It is possible to genetically introduce heterologous protein antigens to the bacterial host that can then be produced and relocated to reside within the OMVs by means of the host secretion mechanisms. Accordingly, in this study we sought to develop a novel platform for recombinant OMV (rOMV) production in the widely used bacterial expression host species, Escherichia coli. Three different lipoprotein signal peptides including their Lol signals and tether sequences-from Neisseria meningitidis fHbp, Leptospira interrogans LipL32, and Campylobactor jejuni JlpA-were combined upstream to the GFPmut2 model protein, resulting in three recombinant plasmids. Pilot expression studies showed that the fusion between fHbp and GFPmut2 was the only promising construct; therefore, we used this construct for large-scale expression. After inducing recombinant protein expression, the nanovesicles were harvested from cell-free culture media by ultrafiltration and ultracentrifugation. Transmission electron microscopy demonstrated that the obtained rOMVs were closed, circular single-membrane particles, 20-200 nm in size. Western blotting confirmed the presence of GFPmut2 in the isolated vesicles. Collectively, although this is a non-optimized, proof-of-concept study, it demonstrates the feasibility of this platform in directing target proteins into the vesicles for OMV-based vaccine development.