• International Journal of Oral Biology
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• v.42 no.2
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• pp.63-70
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• 2017

Selecting an appropriate antigen with optimal immunogenicity and physicochemical properties is a pivotal factor to develop a protein based subunit vaccine. Despite rapid progress in modern molecular cloning and recombinant protein technology, there remains a huge challenge for purifying and using protein antigens rich in hydrophobic domains, such as membrane associated proteins. To overcome current limitations using hydrophobic proteins as vaccine antigens, we adopted in silico analyses which included bioinformatic prediction and sequence-based protein 3D structure modeling, to develop a novel periodontitis subunit vaccine against the outer membrane protein FomA of Fusobacterium nucleatum. To generate an optimal antigen candidate, we predicted hydrophilicity and B cell epitope parameter by querying to web-based databases, and designed a truncated FomA (tFomA) candidate with better solubility and preserved B cell epitopes. The truncated recombinant protein was engineered to expose epitopes on the surface through simulating amino acid sequence-based 3D folding in aqueous environment. The recombinant tFomA was further expressed and purified, and its immunological properties were evaluated. In the mice intranasal vaccination study, tFomA significantly induced antigen-specific IgG and sIgA responses in both systemic and oral-mucosal compartments, respectively. Our results testify that intelligent in silico designing of antigens provide amenable vaccine epitopes from hard-to-manufacture hydrophobic domain rich microbial antigens.

• Journal of Microbiology and Biotechnology
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• v.20 no.11
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• pp.1534-1538
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• 2010

Fed-batch cultures of Hansenula polymorpha were studied to develop an efficient biosystem to produce recombinant human serum albumin (HSA). To comply with this purpose, we used a high-purity oxygen-supplying strategy to increase the viable cell density in a bioreactor and enhance the production of target protein. A mutant strain, H. polymorpha GOT7, was utilized in this study as a host strain in both 5-l and 30-l scale fermentors. To supply high-purity oxygen into a bioreactor, nearly 100% high-purity oxygen from a commercial bomb or higher than 93% oxygen available in situ from a pressure swing adsorption (PSA) oxygen generator was employed. Under the optimal fermentation of H. polymorpha with highpurity oxygen, the final cell densities and produced HSA concentrations were 24.6 g/l and 5.1 g/l in the 5-l fermentor, and 24.8 g/l and 4.5 g/l in the 30-l fermentor, respectively. These were about 2-10 times higher than those obtained in air-based fed-batch fermentations. The discrepancies between the 5-l and 30-l fermentors with air supply were presumably due to the higher contribution of surface aeration over submerged aeration in the 5-l fermentor. This study, therefore, proved the positive effect of high-purity oxygen in enhancing viable cell density as well as target recombinant protein production in microbial fermentations.

• Animal cells and systems
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• v.10 no.3
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• pp.131-135
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• 2006

The ultrastructure of Aspergillus nidulans cell wall in relation to a mannoprotein was studied by scanning and transmission electron microscopy. An mnpAp null-mutant, DMPV1, was used as a negative control of a wild type VER7. To analyze whether the mannoprotein in the cell wall during the development of an mnpAp null-mutant is present or not, immunogold microscopy was also adopted. The surface sculpturing of various cell types - hyphae, conidium, Hulle cell, and ascospore - were not very different between the wild type and the mnpAp-null mutant (DMPV1) as examined by scanning electron microscopy. These results were comparable to those examined by transmission electron microscopy, in that the hyphal cell wall was not indentical between two strains, probably caused by the MnpA protein (MnpAp). MnpAp was absent in both the hyphal cell wall of the DMPV1 strain and the conidial cell wall of a wide type, but clearly recognized in the hyphal cell wall of a wild type.

• Journal of Haehwa Medicine
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• v.13 no.2
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• pp.131-146
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• 2004

In the present study, we exarnined anti-allergic effect of SGBHB in cultured B cells. B cells were prepared from isolated murine splenocytes and activated by co-treatment of anti-CD40 monoclonal antibody and recombinant IL-4 allergens. Anti-allergic effects of SGBHB in activated B cells were determined by measuring B cell surface activated molecules (CD23+ and CD11a+), and expression levels of IL-$1{\beta}$, IL-6, IL-10, TNF-$\alpha$, IgE, and HRF. The major findings are summarized as follows. 1. SGBHB treatment did not produce significant cytotoxic effects on mouse lung fibroblast cells. 2. SGBHB produced significant inhibitory effect on the expression of B cell surface activated molecules (CD23+ and CD11a) in activated B cells. 3. SGBHB treatment significantly inhibited expression levels of IL-$1{\beta}$, IL-6, and TNF-$\alpha$ mRNAs in activated B cells.IL-6 protein levels were significantly decreased by $100{\mu}g/m{\ell}$ of SGBHB treatrrient, and TNF-$\alpha$ protein levels were decreased compared to the control group, but statistically insignificant. 4. SGBHB treatment significantly increased IL-10 at both mRNA and protein levels in activated B cells. 5. SGBHB treatment significantly inhibited levels of IgE production. Thus, the present data suggest that SGBHB has an anti-allergic effect on activated B cells by controlling irnmune responses, and further implicates the possibility on clinical application as a therapeutic agent.

• Journal of Microbiology and Biotechnology
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• v.22 no.2
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• pp.226-229
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• 2012

Bacterial hemoglobin from Vitreoscilla (VHb) is recognized as a good fusion protein for the soluble expression of foreign protein. In this study, we generated a monoclonal antibody (MAb) against VHb for its detection. For the rapid screening of MAb, a protein chip technology based on the Alexa-488 (A488) dye labeling method was introduced. In order to fabricate the chip, the VHb protein was chemically coupled to the chip surface and then the culture supernatants of 84 hybridoma cell lines were spotted onto the VHb chip. The bound MAbs were measured by A488-modified anti-mouse IgG. A single spot (MAb A10) exhibited significantly high signal intensity. The immunoblot analysis evidenced that the MAb A10 can detect VHb-fused proteins with high specificity.

• The Korean Journal of Zoology
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• v.38 no.4
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• pp.457-464
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• 1995

Hela cells, a transformed human epithelial cell line, attach to various substrata but subsequent spreading is specific to collagen or gelatin. The spreading is initiated by the activation of phospholipase $A_2$ (PLA$_2$) which produces arachidonic acid (AA) as a consequence of cell surface collagen receptor clustering. This study examines the mechanism of PLA$_2$activation and which phospholipids are hydrolyzed by PIA$_2$ to release AA in response to Hela cell adhesion to a gelatin substratum. The levels of phosphatidyicholine decreases, among various phospholipids, during attachment and spreading of Hela cells. Lysophosphatidyicholine Is the only lysophospholipids formed during ileLa cell adhesion indicating that clustered collagen receptors activate PLA$_2$to hydrolyze posphatidylcholine to AA and lysophosphatidylcholine. Among various molecular entitles which are known to regulate PLA$_2$ activation, we have previously shown that PLA2 activation is not mediated by either changes in $Ca_2$+ levels, alkalinization of cytoplasmic p11, or activation of protein hinase C. It is also likely that PIA2 activation is not mediated by either pertussis or cholera toxinsensitive G proteins as those toxins do not affect both AA release and cell spreading.

• BMB Reports
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• v.40 no.5
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• pp.832-838
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• 2007

A novel inhibitory protein against blood coagulation factor Va (FVa) was purified from muscle protein of granulated ark (Tegillarca granosa, order Arcoida, marine bivalvia) by consecutive FPLC method using anion exchange and gel permeation chromatography. In the results of ESI-QTOF tandem mass analysis and database research, it was revealed that the purified T. granosa anticoagulant protein (TGAP) has 7.7 kDa of molecular mass and its partial sequence, HTHLQRAPHPNALGYHGK, has a high identity (64%) with serine/threonine kinase derived from Rhodopirellula baltica (order Planctomycetales, marine bacteria). TGAP could potently prolong thrombin time (TT), corresponding to inhibition of thrombin (FIIa) formation. Specific factor inhibitory assay showed that TGAP inhibits FVa among the major components of prothrombinase complex. In vitro assay for direct-binding affinity using surface plasmon resonance (SPR) spectrometer indicated that TGAP could be directly bound with FVa. In addition, the binding affinity of FVa to FII was decreased by addition of TGAP in dose-dependant manner ($IC_{50}$ value = 77.9 nM). These results illustrated that TGAP might interact with a heavy chain of FVa ($FVa_H$) bound to FII in prothrombin complex. The present study elucidated that non-cytotoxic T. granosa anticoagulant protein (TGAP) bound to FVa can prolong blood coagulation time by inhibiting conversion of FII to FIIa in blood coagulation cascade. In addition, TGAP did not significantly (P < 0.05) show fibrinolytic activity and cytotoxicity on venous endothelial cell line (ECV 304).

• KSBB Journal
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• v.22 no.6
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• pp.393-400
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• 2007

Analysis of protein interactions/functions in a microarray format has been of great potential in drug discovery, diagnostics, and cell biology, because it is amenable to large-scale and high-throughput biological assays in a rapid and economical way. In recent years, the protein microarray have broaden their utility towards the global analysis of protein interactions on a proteome scale, the functional activity analysis based on protein interactions and post-translational modifications (PTMs), and the discovery of biomarkers through profiling of protein expression between sample and reference pool. As a promising tool for proteomics, the protein microarray technology has advanced outstandingly over the past decade in terms of surface chemistry, acquisition of relevant proteins on a proteomic level, and detection methods. In this article, we briefly describe various techniques for development of protein microarray, and introduce developmental state of protein microarray and its applications.

• Korean Journal of Microbiology
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• v.48 no.3
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• pp.187-191
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• 2012

Two-component system (TCS)-based bacterial zinc and copper biosensors, in which green fluorescent protein (GFP) is expressed under the control of zraP and cusC promoter in ZraS/R and CusS/R TCS, were evaluated in artificial wastewater. Bacterial biosensors developed in this study efficiently expressed GFP by the recognition of $Zn^{2+}$ and $Cu^{2+}$ in artificial wastewater. Secondly, TCS-based zinc and copper removing bacteria with the peptide displayed on cell surface were examined in artificial wastewater. Zinc and copper removing bacteria expressed the peptide as a fusion protein such as OmpC-ZBP (zinc binding peptide) and OmpC-CBP (copper binding peptide) on the cell surface when sensing exogenous $Zn^{2+}$ and $Cu^{2+}$ through ZraS/R and CusS/R TCS. The recombinant cell expressing metal-adsorbing peptide could efficiently remove copper and zinc (15 and 18 mg/g dry cell weight, respectively) in artificial wastewater. Therefore, it was demonstrated that the TCS-based recombinant cell for the recognition or removal of heavy metal functions well in artificial wastewater environment.

• BMB Reports
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• v.35 no.4
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• pp.395-402
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• 2002

Caveolae are small, flask-shaped, non-clathrin coated invaginations of the plasma membrane of many mammalian cells. Caveolae have a coat that includes caveolin. They have been implicated in numerous cellular processes, including potocytosis. Since the human folate receptor (hFR) and other glycosyl-phosphatidylinositol (GPI)-tailed proteins have been co-localized to caveolae, we studied the caveolin role in the hFR function by transfecting hFR and/or caveolin cDNA into Fischer rat thyroid epithelial (FRT) cells that normally do not express detectable levels of either protein. We isolated and characterized stable clones as follows: they express (1) high levels of caveolin alone, (2) hFR and caveolin, or (3) hFR alone. We discovered that hFR is correctly processed, sorted, and anchored by a GPI tail to the plasma membrane in FRT cells. No difference in the total folic acid binding or cell surface folic acid binding activity were found between the FRT cells that were transfected with hFR, or cells that were transfected with hFR and caveolin. The hFR that was expressed on the cell surface of clones that were transfected with hFR was also sensitive to phosphatidylinositol-specific phospholipase C (PI-PLC) release, and incorporated radiolabeled ethanolamine that supports the attachment of a GPI-tail on hFR. We conclude that the processing, sorting, and function of hFR is independent on the caveolin expression in FRT cells.