• Molecules and Cells
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• v.39 no.6
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• pp.460-467
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• 2016

Bacteriophytochromes are phytochrome-like light-sensing photoreceptors that use biliverdin as a chromophore. To study the biochemical properties of the Deinococcus radiodurans bacteriophytochrome (DrBphP) protein, two anti-DrBphP mouse monoclonal antibodies (2B8 and 3H7) were generated. Their specific epitopes were identified in our previous report. We present here fine epitope mapping of these two antibodies by using truncation and substitution of original epitope sequences in order to identify minimized epitope peptides. The previously reported original epitope sequences for 2B8 and 3H7 were truncated from both sides. Our analysis showed that the minimal peptide sequence lengths for 2B8 and 3H7 antibodies were nine amino acids (RDPLPFFPP) and six amino acids (PGEIEE), respectively. We further characterized these peptides in order to investigate their reactivity after single deletion and single substitution of the original peptides. We found that single-substituted 2B8 epitope (RDPLPAFPP) and dual-substituted 3H7 epitope (PGEIAD) showed significantly increased reactivity. These two antibodies with high reactivity for the short modified peptide sequences are valueble for developing new peptide tags for protein research.

• Biomedical Science Letters
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• v.7 no.3
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• pp.103-110
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• 2001

$\alpha$-Fetoprotein(AFP) is a good marker for the detection of several diseases such as hepatocellular carcinoma, gonadal germ cell tumor, gastric tumor, and Down's syndrome. In this study, we developed ELISA, using synthetic peptides corresponding to the epitopes of AFP. Five kinds of peptides were synthesized from AFP to produce antibodies in rats that recognize AFP in human plasma as well as amniotic fluid and do not cross-react with serum albumin. All five kinds of antibodies showed good reactivities with their peptide-keyhole limpet hemocyanin conjugates. Anti-synthetic peptide 1 (R-N-E-Y-G-I-A-S-I-L, 4-13) antibody, in particular, reacted well with AEP as well as synthetic peptide 1-KLH but not with human serum albumin. The binding affinity(Kd) was 2.7$\times$10$^{-9}$M for peptide 1 and 6.8$\times$10$^{-8}$M for AEP. The range for measurement of AFP was 10~1,000 ng/ml. The within-assay and between-assay coefficients of variance(CV) were 4.83% and 10.97%, respectively. In a sample of 31 sera and 33 amniotic fluids, there was a good correlation between AFP values determined in this assay and those in a commercial kit. These results indicate that the antibodies against synthetic peptides corresponding to the epitopes of AFP are highly specific to APP and synthetic peptide-based ELISA would be useful for the measurement of human AFP.

• Microbiology and Biotechnology Letters
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• v.35 no.2
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• pp.112-117
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• 2007

Protein kinases play very important role for maintaining viability in prokaryote and eukaryote. The metabolism of prokaryotic cell is generally regulated by bacterial two-component regulatory systems that are composed of histidine and asparitic acid kinases, however, some eukaryotic signal transduction system such as, serine and threonine kinases, have been also found to be involved in the regulation of morphogenesis and physiological differentiation in Streptomyces. Streptomyces griseus, a streptomycin producer, was expected to have varlous types of eukaryotic-type serine/threonine protein kinases, controlling morphogenesis. Thus, many steps of chromatographies were applied to isolate serine and threonine kinases from S. griseus IFO13350. The immunoaffinity steps using anti-phosphoserine, anti-phosphothreonine, and anti-phosphotyrosine agarose column chramatographies were successfully introduced to identify eukaryotic protein kinases from S. griseus IFO13350. Eight proteins with the expected molecular weight of 14, 29, 31, 35, 40, 52, 56, and 60 kDa, were identified on SDS-PAGE, and the their kination activity was confirmed by nonradioactive protein kination assay using FITC-labeled peptide as the substrate.

• Proceedings of the Microbiological Society of Korea Conference
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• 2002.10a
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• pp.121-124
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• 2002

This work was initiated to develope a recombinant oral poliovaccine (OPV), which is highly advanced in safety (minimizing VAPP) by introducing Type 2,3 poliovirus epitopes into our RPS-Vax system. We have introduced several potential vaccine epitopes of poliovirus Type 2, and 3 into RPS-Vax system, resulting in production of recombinant polioviruses. Any of these chimeric viruses, however, were not detected for their foreign gene expression by serotype-specific mouse antiserum. We have designed several folding units to stabilize the introduced vaccine protein and attached short epitope-concatamer or epitope-multimer to them, followed by production of chimeric viruses. Only those who have an HIV-1 Tat-mediated folding unit were nicely detected for the introduced foreign proteins by anti-Tat antiserum and type-specific peptide-induced antisera. Nevertheless, introduced epitopes were not detected in Western blot experiment with each serotype-specific antiserum. None of the mice inoculated with these chimeric viruses showed preventative immunity when challenged with Lansing and Leon wildtype 2 and 3 poliovirus, and the antiserum did not show neutralizing capacity in vitro. Conformational epitope covering B/C loop region of type 2 and 3 were newly designed by computer modeling, and introduced into the RPS-Vax vector system, followed by production of chimeric viruses. Introduced epitope regions were nicely detected by anti-Tag23 mAb or peptide antibody, but still not detected by poliovirus antiserum. Nevertheless, neutralizing antibody was detected in the Tg-PVR mice even when inoculated once with these chimeric viruses. Also, the immunized mice showed perfect preventative immunity against the wild Type poliovirus Lancing or Leon. When boosted appropriately, those chimeric virus-inoculated Tg-PVR mice produced equivalent amounts of neutralizing antibody to those in Sabin 2/3-immunized mice. These data strongly suggest that our recombinant poliovirus (RPS-PV2 and RPS-PV3) can be used as a safe and effective rec-OPV instead of any preexisting poliovaccine.

• Journal of Periodontal and Implant Science
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• v.41 no.2
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• pp.54-59
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• 2011

Purpose: The aim of this study was to define the immunoreactive specificity of Porphyromonas gingivalis (P. gingivalis) heat shock protein (HSP) 60 in periodontitis and atherosclerosis. Methods: In an attempt to define the cross-reactive bacterial heat-shock protein with human self-antigen at molecular level, we have introduced a novel strategy for cloning hybridoma producing anti-P. gingivalis HSP 60 which is polyreactive to bacterial HSPs or to the human homolog. Results: Five cross-reactive clones were obtained which recognized the #19 peptide (TLVVNRLRGSLKICAVKAPG) among 37 synthetic peptides (20-mer, 5 amino acids overlapping) spanning the whole molecule of P. gingivalis HSP 60. We have also established three anti-P. gingivalis HSP 60 monoclonal antibodies demonstrating mono-specificity. These clones recognized the #29 peptide (TVPGGGTTYIRAIAALEGLK). Conclusions: Peptide #19 and #29 of P. gingivalis HSP 60 might be important immunoreactive epitopes in the immuno-pathogenic mechanism of bacterial antigen-triggered autoimmune diseases.

• Korean Journal of Clinical Laboratory Science
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• v.39 no.1
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• pp.42-48
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• 2007

This study was carried out to review and evaluate anti-CCP ELISA assay for diagnostic in RA patients from an early arthritis clinic (EAC). The subjects were obtained from patients visiting the outpatient clinic of the Dept. of Rhumatology med.of P hospital in Daegu, during 6 months from July 1, 2006 to December 31, 2006. The subjects were 140 cases : 80 cases from RA patients (60 women and 20 men; mean age 58 years; range, 32-68 years) confirmed by clinical diagnostic. 39 cases of these RA patients were classified as having early RA (EAC). 50 cases (non-RA) did not fulfill the criteria for RA, and 10 cases were from healthy individuals. We performed the analysis with solid phase-ELISA method (ETI-max3000, Diasorin; Italy) for anti-CCP and Nephelometry assay (Roche/Hitachi 902 analyzer; USA) for RF. The results obtained were summarized as follows ; anti-CCP ELISA is more specific than RF Nephelometry assay (specificity 94% vs 90%) to diagnose RA patients with suspected EAC (early arthritis clinic). The combination test "anti-CCP and RF" had a very high specificity (specificity 98.3%, PPV; RA group 96%, EAC 95%), the difference was statistically significant (p<0.05). Anti-CCP ELISA had more sensitivity in EAC (Early arthritis clinic) patients than chronic RA patients (sensitivity 64% vs 24%, respectively), anti-CCP of RA group and EAC group was more specific than RF (anti-CCP PPV; 92%, 89% vs 89%, 81% respectively), the difference was statistically significant (p<0.05). The difference of antibody concetration between anti-CCP and RF for RA and the control group is statistically significant (p<0.05). In conclusion, anti-CCP ELISA testing may be useful if performed concomitantly with RF Nephelometry assay to diagnose RA patients with suspected EAC (early arthritis clinic).

• BMB Reports
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• v.40 no.4
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• pp.562-570
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• 2007

Soluble or cell-bound IL-1 receptor accessory protein (IL-1RAcP) does not bind IL-1 but rather forms a complex with IL-1 and IL-1 receptor type I (IL-1RI) resulting in signal transduction. Synthetic peptides to various regions in the Ig-like domains of IL-1RAcP were used to produce antibodies and these antibodies were affinity-purified using the respective antigens. An anti-peptide-4 antibody which targets domain III inhibited 70% of IL-$1\beta$-induced productions of IL-6 and PGE2 from 3T3-L1 cells. Anti-peptide-2 or 3 also inhibited IL-1-induced IL-6 production by 30%. However, antipeptide-1 which is directed against domain I had no effect. The antibody was more effective against IL-$1\beta$ compared to IL-$1\alpha$. IL-1-induced IL-6 production was augmented by coincubation with PGE2. The COX inhibitor ibuprofen blocked IL-1-induced IL-6 and PGE2 production. These results confirm that IL-1RAcP is essential for IL-1 signaling and that increased production of IL-6 by IL-1 needs the co-induction of PGE2. However, the effect of PGE2 is independent of expressions of IL-1RI and IL-1RAcP. Our data suggest that domain III of IL-1RAcP may be involved in the formation or stabilization of the IL-1RI/IL-1 complex by binding to epitopes on domain III of the IL-1RI created following IL-1 binding to the IL-1RI.

• IMMUNE NETWORK
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• v.1 no.1
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• pp.77-86
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• 2001

Background: Hepatitis C virus(HCV), a family of Flaviviridae, has a host cell-derived envelope containing a positive-stranded RNA genome, and has been known as the maj or etiological agent for chronic hepatitis, hepatic cirrhosis, and hepatocellular carcinoma. There remains a need to dissect a molecular mechanism of pathogenesis for the development of therapeutic and effective preventive measure for HCV. Identification of cellular receptor is of central importance not only to understand the viral pathogenesis, but also to exploit strategies for prevention of HCV. This study was aimed at identifying peptide mimotopes inhibiting the binding of E2 protein of HCV to MOLT-4 cell. Methods: In this study, phage peptide library displaying a random peptides consisting of 7 or 12 random peptides was employed in order to pan against E2 protein. Free HCV particles were separated from the immune complex forms by immunoprecipitation using anti-human IgG antibody, and used for HCV-capture ELISA. To identify the peptides inhibiting E2-binding to MOLT-4 cells, E2 protein was subj ect to bind to MOLT-4 cells under the competition with phage peptides. Results: Several phage peptides were selected for their specific binding to E2 protein, which showed the conserved sequence of SHFWRAP from 3 different peptide sequences. They were also able to recognize the HCV particles in the sera of HCV patients captured by monoclonal antibody against E2 protein. Two of them, showing peptide sequence of HLGPWMSHWFQR and WAPPLERSSLFY respectively, were revealed to inhibit the binding of E2 protein to MOLT-4 cell efficiently in dose dependent mode. However, few membrane-associated receptor candidates were seen using Fasta3 programe for homology search with these peptides. Conclusion: Phage peptides containing HLGPWMSHWFQR and WAPPLERSSLFY respectively, showed the inhibition of E2-binding to MOLT-4 cells. However, they did not reveal any homologues to cellular receptors from GenBank database. In further study, cellular receptor could be identified through the screening of cDNA library from MOLT-4 or hepatocytes using antibodies against these peptide mimotopes.

• Biotechnology and Bioprocess Engineering:BBE
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• v.3 no.2
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• pp.82-86
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• 1998

The maltose binding protein (MBP) fusion protein system is a versatile tool to express and isolate recombinant proteins in E. coli. In this system, MBP fusion proteins are efficiently isolated from whole cell lysate using amylose conjugated agarose beads and then eluted by competition with free maltose. Since MBP is a rather large molecule (∼42 kDa), for further experiments, the MBP part is usually proteolytically cleaved from the fusion protein and subsequently removed by ion-exchange chromatography or rebinding to amylose columns after washing out excess and MBP-bound maltose. In the present study, we have developed an improved method for the removal of cleaved MBP, which is advantageous over conventional methods. In this method, factor Xa cleaved MBP fusion proteins were incubated with Sepharose beads conjugated with MBP specific monoclonal antibodies and then precipitated buy centrifugation, resulting in highly purified proteins in the supernatant.

• Journal of Ginseng Research
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• v.48 no.1
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• pp.59-67
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• 2024

Background: Alzheimer's disease (AD) has memory impairment associated with aggregation of amyloid plaques and neurofibrillary tangles in the brain. Although anti-amyloid β (Aβ) protein antibody and chemical drugs can be prescribed in the clinic, they show adverse effects or low effectiveness. Therefore, the development of a new drug is necessarily needed. We focused on the cognitive function of Panax ginseng and tried to find active ingredient(s). We isolated panaxcerol D, a kind of glycosyl glyceride, from the non-saponin fraction of P. ginseng extract. Methods: We explored effects of acute or sub-chronic administration of panaxcerol D on cognitive function in scopolamine- or Aβ_25-35 peptide-treated mice measured by several behavioral tests. After behavioral tests, we tried to unveil the underlying mechanism of panaxcerol D on its cognitive function by Western blotting. Results: We found that pananxcerol D reversed short-term, long-term and object recognition memory impairments. The decreased extracellular signal-regulated kinases (ERK) or Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) in scopolamine-treated mice was normalized by acute administration of panaxcerol D. Glial fibrillary acidic protein (GFAP), caspase 3, NF-kB p65, synaptophysin and brainderived neurotrophic factor (BDNF) expression levels in Aβ_25-35 peptide-treated mice were modulated by sub-chronic administration of panaxcerol D. Conclusion: Pananxcerol D could improve memory impairments caused by cholinergic blockade or Aβ accumulation through increased phosphorylation level of ERK or its anti-inflammatory effect. Thus, panaxcerol D as one of non-saponin compounds could be used as an active ingredient of P. ginseng for improving cognitive function.