• Microbiology and Biotechnology Letters
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• v.25 no.2
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• pp.129-136
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• 1997

Helicobacter pylori is a spiral-shaped, microaerophilic human gastric pathogen causing chronic-active gastritis in association with duodenal ulcer and gastric cancer. To investigate the possibility of H. pylori outer membrane proteins (OMPS) as the oral vaccine antigens, sarcosine-insoluble outer membrane fraction has been prepared from H. pylori NCTC 11637. The major OMPs having apparent molecular masses of 62 kDa, 54 kDa and 33 kDa were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which were identified as urease B subunit (UreB), heat shock protein (Hsp54 kDa) and urease A subunit (UreA), respectively. Minor protein bands of 57 kDa, 52 kDa, 40 kDa, 36 kDa and 31 kDa were also observed. The antigenicity of H. pylori OMPs and antigenic cross-reactivity among the strains were determined by immunoblot analysis using anti-H. pylori OMPs antisera or intestinal lavage solutions. The results showed that UreB, Hsp54 kDa, UreA and 40 kDa proteins vigorously stimulated mucosal immune response rather than systemic immunity. From this results, these proteins seemed to be useful as the antigen candidates for the oral vaccine. The immunoblotting results with surface proteins from eight isolated H. pylori strains were similar to that of H. pylori NCTC 11637. The IgA which had been arised from oral administration of H. pylori OMPs, was able to bind H. pylori whole-cells.

• Journal of Microbiology and Biotechnology
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• v.23 no.5
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• pp.587-601
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• 2013

During the last few decades, it has become evident that the compatibility of the yeast biochemical environment with the ability to process and translate the RNA transcript, along with its capacity to modify a translated protein, are relevant requirements for selecting this host cell for protein expression in several pharmaceutical and clinical applications. In particular, Pichia pastoris is used as an industrial host for recombinant protein and metabolite production, showing a powerful capacity to meet required biomolecular target production levels in high-throughput assays for functional genomics and drug screening. In addition, there is a great advantage to using P. pastoris for protein secretion, even at high molecular weights, since the recovery and purification steps are simplified owing to relatively low levels of endogenous proteins in the extracellular medium. Clearly, no single microexpression system can provide all of the desired properties for human protein production. Moreover, chemical and physical bioprocess parameters, including culture medium formulation, temperature, pH, agitation, aeration rates, induction, and feeding strategies, can highly influence product yield and quality. In order to benefit from the currently available wide range of biosynthesis strategies using P. pastoris, this mini review focuses on the developments and technological fermentation achievements, providing both a comparative and an overall integration analysis. The main aim is to highlight the relevance and versatility of the P. pastoris biosystem to the design of more cost-effective microfactories to meet the increasing demands for recombinant membrane proteins and clinical antibodies for several therapeutic applications.

• Korean Journal of Microbiology
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• v.47 no.1
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• pp.7-13
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• 2011

In general, expression of membrane protein in Escherichia coli is very toxic to the host organism, but the mechanism for the toxicity is not clear yet. Expression of human purinergic receptor $P2X_4$ was found to be extremely toxic to the host E. coli. We examined this toxicity by isolation and analysis of less toxic mutant proteins. We could isolate 30 less toxic mutants of $P2X_4$ after hydroxylamine mutagenesis. Western blot showed that all of them produced proteins smaller than the wild type $P2X_4$. DNA sequencing of two largest mutant proteins showed that they were lost its second transmembrane domain. Localization analysis of these mutant proteins showed that they are not in cytoplasmic membrane, but in inclusion bodies. These data showed that inactive truncated $P2X_4$ is not toxic to E. coli and membrane integration and functionality of $P2X_4$ may be needed to show host toxicity.

• Journal of the Korean Magnetic Resonance Society
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• v.14 no.2
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• pp.88-104
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• 2010

Human melanocortin-4 receptor (hMC4R) has a critical role in part of energy homeostasis, and their heterozygous mutations related in genetic cause of severe human obesity. In order to study the structure and function of these membrane proteins, it is important to prepare the samples. However, the preparation of transmembrane peptide is seriously difficult and time-consuming. Overexpression and purification of membrane proteins was reported to be difficult due to their innate insoluble and toxic properties. Among the many difficulties, the most important is the difficulty in obtaining sufficient quantities of purified protein. Recently, we succeed to produce large amounts of the second transmembrane domain from the wild-type hMC4R (wt-TM2) and D90N mutant hMC4R (m-TM2) and proposed the structural difference of them in membrane-like environments. In this paper, we demonstrate the optimization procedures to express and purify wt-TM2 or m-TM2 peptides, and solution NMR studies in different detergents to get high-resolution spectra were also described.

• Journal of Plant Biology
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• v.33 no.4
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• pp.325-332
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• 1990

Light membrane vesicles were isolated from the zucchini hypocotyl by floatation on ficoll density gradients and the proteins were solubilized with Triton X100. Three ATP-hydrolyzing enzymes were partially purified by ion-exchange and gel filtration chromatography and isoelectric focusing. There are plasma membrane-type ATPase whose activity was inhibited by vanadate but not by nitrate, tonoplast-type ATPase which was sensitive to nitrate but insensitive to vanadate and one having a phosphatase activity with a pI value different from that of an acid phosphatase. A fraction was obtained after DEAE-ion-exchange chromatography crossreacting with polyclonal antibodies against Ca2+ -ATPase from human erythrocytes.

• Parasites, Hosts and Diseases
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• v.45 no.4
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• pp.287-293
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• 2007

The identification and characterization of antigens that elicit human T cell responses is an important step toward understanding of Leishmania major infection and ultimately in the development of a vaccine. Micropreparative SDS-PAGE followed by electro transfer to a PVDF membrane and elution of proteins from the PVDF, was used to separate 2 novel proteins from L. major promastigotes, which can induce antibodies of the IgG2a isotype in mice and also are recognized by antisera of recovered human cutaneous leishmaniasis subjects. Fractionation of the crude extract of L. major revealed that all detectable proteins of interest were present within the soluble Leishmania antigens (SLA). Quantitation of these proteins showed that their expression in promastigotes is relatively very low. Considering the molecular weight, immunoreactivity, chromatographic and electrophoretic behavior in reducing and non-reducing conditions, these proteins are probably 2 isoforms of a single protein. A digest of these proteins was resolved on Tricine-SDS-PAGE and immunoreactive fragments were identified by human sera. Two immunoreactive fragments (36.4 and 34.8 kDa) were only generated by endoproteinase Glu-C treatment. These immunoreactive fragments or their parent molecules may be ideal candidates for incorporation in a cocktail vaccine against cutaneous leishmaniasis.

• Journal of Microbiology and Biotechnology
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• v.19 no.9
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• pp.1034-1040
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• 2009

Porin proteins of Gram-negative bacteria are outer membrane proteins that act as receptors for bacteriophages and are involved in a variety of functions like solute transport, pathogenesis, and immunity. Salmonella enterica serovar Typhi (S. Typhi), a Gram-negative bacterium, is the causative agent of typhoid fever. Porins of S. Typhi have been shown to have a potential role in diagnostics and vaccination. In the present study, the major outer membrane proteins OmpF and OmpC from S. Typhi were cloned in pQE30UA vector and expressed in E. coli. The immunogenic nature of the recombinant porin proteins were evaluated by ELISA by raising hyperimmune sera in Swiss Albino mice with three different adjuvants (i.e., Freund's adjuvant and two human-compatible adjuvants like montanide and aluminium hydroxide gel) and proved to be immunogenic. The recombinant OmpF and OmpC generated in this work may be used for further studies for vaccination and diagnostics.

• Journal of Oral Medicine and Pain
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• v.24 no.3
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• pp.235-244
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• 1999

The present study was performed to investigate the binding between salivary proteins(low-molecular-weight mucin;MG2, amylase, proline-rich proteins;PRPs) and oral pathogens(Streptococcus gordonii, Actinomyces viscosus, Staphylococcus aureus) by using solid-phase assay. In the case of transferring proteins to Immobilon-P, S. gordonii binds to MG2. A. viscosus binds to MG2, amylase, and PRPs, and S. aureus binds to MG2 and amylase. On nitrocellulose membrane, S, gordonii and A. viscosus bind to MG2, amylase, and PRPs. S. aureus binds to MG2 and PRPs. However, rabbit anti-A. viscosus antisera and rabbit anti-S. aureus antisera showed cross reactivity to PRPs adsorbed to only nitrocellulose membrane in negative control experiments, which were done without bacterial overlay. The results were different according to the membrane used as solid-phase, which reflected the assay-sensitive nature of binding experiment. PRPs and amylase are known to be components of tooth enamel pellicle. In addition, there was experimental evidence that PRPs and MG2 may covalently bind to oral mucosal epithelium. Considering above facts, the results of the present study can provide information on the interactions between salivary proteins and oral bacteria on tooth and oral mucosal surfaces.

• Parasites, Hosts and Diseases
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• v.49 no.1
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• pp.79-83
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• 2011

Trichomoniasis is a sexually transmitted disease due to infection with Trichomonas vaginalis, and it can cause serious consequences for women's health. To study the virulence factors of this pathogen, T. vaginalis surface proteins were investigated using polyclonal antibodies specific to the membrane fractions of T. vaginalis. The T. vaginalis expression library was constructed by cloning the cDNA derived from mRNA of T. vaginalis into a phage ${\lambda}$ Uni-ZAP XR vector, and then used for immunoscreening with the anti-membrane proteins of T. vaginalis antibodies. The immunoreactive proteins identified included adhesion protein AP65-1, ${\alpha$-actinin, kinesin-associated protein, teneurin, and 2 independent hypothetical proteins. Immunofluorescence assays showed that AP65-1, one of the identified immunogenic clones, is prevalent in the whole body of T. vaginalis. This study led us to identify T. vaginalis proteins which may stimulate immune responses by human cells.

• Journal of Microbiology and Biotechnology
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• v.13 no.3
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• pp.444-450
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• 2003

In order to develop an effective means to treat P. aeruginosa infections, we have purified P. aeruginosa outer membrane proteins (OMPs)-specific human IgG antibody. In this study, we investigated the protective activity of the purified anti-OMPs IgC against P. aeruginosa infection in a rabbit endophthalmitis model. Rabbits were inoculated by an intravitreal injection with P. aeruginosa, and treated with a single dose of 1 mg anti-P. aeruginosa OMPs IgG. All the control rabbits predominantly developed edematous responses and opacity in the eyes, but the rabbits treated with the antibody showed only very limited degree of edema. Aliquots of the vitreous humor were extracted and analyzed for the number of viable bacteria and endotoxin level. The results showed that the anti-OMPs IgC significantly reduced the bacterial count compared with the control group, and that the endotoxin level of the vitreous from the IgG-treated rabbits was more than 70-fold lower 6 h after the administration than the control animals. These data suggested that the anti-P. aeruginosa OMPs IgG is effective in inhibiting the bacterial growth and thereby in reducing endotoxin levels in the vitreous, warranting further development of the anti-P. aeruginosa OMPs IgG as a therapeutic means for treating Pseudomonas endophthalmitis.