• The Korean Journal of Food And Nutrition
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• v.9 no.4
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• pp.452-458
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• 1996

A PPIase gene of Bacillus stearothermophilus was screened from a genomic library by plaque hybridization using the A-1 primer as a probe. A PPIase positive plaque contained a 3.0kb insert of the chromosomal DNA. A 3.0kb fragment was subcloned into pUC18, resulting pPI1-40. A DNA fragment encoding the N-terminal portion of the PPIase in pPi-40 was amplified by polymerase chain reaction(PCR) method using the A-1 and B-2 primers. The amplified fragment was cloned into the Sma I site of pUC18 and recombinant plasmid was designated as pSN-18. The nucleotide sequence of 167bp fragment was determined. The deduced amino acid sequence of PPIase was completely matched with the determined N-terminal amino acid sequence of PPIase B. stearothermophilus. The translated protein sequence of PPIase B. stearothermophilus was compared with sequence from periplasmic PPIase from Escherichina coil ; homogies of 16 and 58%, respectively, were found. The clond PPIase gene was over-expressed in E. coil cell using pUC19 as an expression vector. The enzyme was partially purified by heat treatment and colum chromatochraphy on DEAE-Sepharose CL-6B. The molecular weight of the enzyme was dermined to be about 18.0 kDal by SDS-PAGE.

• Molecules and Cells
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• v.38 no.4
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• pp.373-379
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• 2015

Pyruvate kinase M2 isoform (PKM2), a rate-limiting enzyme in the final step of glycolysis, is known to be associated with the metabolic rewiring of cancer cells, and considered an important cancer therapeutic target. Herein, we report a novel PKM2 activator, PA-12, which was identified via the molecular docking-based virtual screening. We demonstrate that PA-12 stimulates the pyruvate kinase activity of recombinant PKM2 in vitro, with a half-maximal activity concentration of $4.92{\mu}M$, and effectively suppresses both anchorage-dependent and -independent growth of lung cancer cells in non-essential amino acid-depleted medium. In addition, PA-12 blocked the nuclear translocalization of PKM2 in lung cancer cells, resulting in the inhibition of hypoxia response element (HRE)-mediated reporter activity as well as hypoxia-inducible factor 1 (HIF-1) target gene expression, eventually leading to the suppression of cell viability under hypoxia. We also verified that the effects of PA-12 were dependent on PKM2 expression in cancer cells, demonstrating the specificity of PA-12 for PKM2 protein. Taken together, our data suggest that PA-12 is a novel and potent PKM2 activator that has therapeutic implications for lung cancer.

• Journal of Microbiology and Biotechnology
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• v.25 no.4
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• pp.537-546
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• 2015

Classical swine fever (CSF) is a highly contagious disease of pigs caused by CSF virus (CSFV). E2 is the major viral envelope protein of immune dominance that induces neutralizing antibodies and confers protection against CSFV infection. The B/C domains of E2 are variable among CSFV isolates, which could affect immunogenicity and binding to antibodies. We attempted to characterize the epitope recognized by a monoclonal antibody 2B6 (mAb-2B6) raised against the E2 B/C domains of the vaccine C-strain and to examine if mutations in the epitope region would affect antibody binding and viral neutralization. The epitope specific for mAb-2B6 recognition is linear, spanning five residues ⁷⁷⁴DGXNP⁷⁷⁸ in the B/C domains. The residue N777 is indispensable for the specificity. The epitope exists only in group 1 strains, but not in those of group 2. The recombinant viruses containing individual mutations on the epitope region lost the reactivity to mAb-2B6. The mutant virus RecC-N777S had low replication potential, about 10-fold decrease in the yield of progeny virus particles, whereas the mutant virus RecC-P778A reverted to proline upon continuous passaging. The mutations on the mAb-2B6 epitope region did not affect neutralization by anti-C-strain polyclonal sera from pigs. Deletion from aa774 covering the mAb-2B6 epitope, but not that from aa781, also affected binding with the polyclonal antibodies from vaccinated pigs, although the major binding region for the vaccinated antibodies is aa690-773.

• Journal of Microbiology and Biotechnology
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• v.22 no.3
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• pp.292-300
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• 2012

We report the expression, purification, and characterization of L-asparaginase (AnsA) from Rhizobium etli. The enzyme was purified to homogeneity in a single-step procedure involving affinity chromatography, and the kinetic parameters $K_m$, $V_{max}$, and $k_{cat}$ for L-asparagine were determined. The enzymatic activity in the presence of a number of substrates and metal ions was investigated. The molecular mass of the enzyme was 47 kDa by SDS-PAGE. The enzyme showed a maximal activity at $50^{\circ}C$, but the optimal temperature of activity was $37^{\circ}C$. It also showed maximal and optimal activities at pH 9.0. The values of $K_m$, $V_{max}$, $k_{cat}$, and $k_{cat}/K_m$ were $8.9{\pm}0.967{\times}10^{-3}$ M, $128{\pm}2.8$ U/mg protein, $106{\pm}2s^{-1}$, and $1.2{\pm}0.105{\times}10^4M^{-1}s^{-1}$, respectively. The L-asparaginase activity was reduced in the presence of $Mn^{2+}$, $Zn^{2+}$, $Ca^{2+}$, and $Mg^{2+}$ metal ions for about 52% to 31%. In addition, we found that $NH_4{^+}$, L-Asp, D-Asn, and ${\beta}$-aspartyl-hydroxamate in the reaction buffer reduced the activity of the enzyme, whereas L-Gln did not modify its enzymatic activity. This is the first report on the expression and characterization of the L-asparaginase (AnsA) from R. etli. Phylogenetic analysis of asparaginases reveals an increasing group of known sequences of the Rhizobial-type asparaginase II family.

• IMMUNE NETWORK
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• v.14 no.3
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• pp.164-170
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• 2014

JL1, a specific epitope on CD43, is a potential biomarker for the diagnosis of acute leukemia. Although qualitative assays for detecting leukemia-specific CD43 exist, there is a need to develop quantitative assays for the same. Here, we developed two novel monoclonal antibodies (mAbs), 2C8 and 8E10, recognizing different epitopes on CD43. These clones are capable of pairing with YG5, another mAb against JL1 epitope, because they were selectively obtained using sandwich ELISA. Antigens recognized by 2C8 and 8E10 were confirmed as CD43 by western blotting using the CD43-hFC recombinant protein. When expression on various leukemic cell lines was investigated, 2C8 and 8E10 displayed a disparity in the distribution of the epitope. Enzyme assays revealed that these mAbs recognized a sialic acid-dependent epitope on CD43. Using normal thymus and lymph node paraffin-embedded tissues, we confirmed a difference in the epitopes recognized by the two mAbs that was predicted based on the maturity of the cells in the tissue. In summary, we developed and characterized two mAbs, 2C8 and 8E10, which can be used with YG5 in a sandwich ELISA for detecting leukemia-specific CD43.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.34 no.4
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• pp.419-427
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• 2008

The present study aimed to investigate the osteogenic potentials of differentiated osteoblast-like cells (DOCs) induced from bone marrow-derived mesenchymal stem cells (MSCs) on ${\beta}-tricalcium$ phosphate (${\beta}-TCP$) with recombinant human bone morphogenetic protein (rhBMP-2) in vitro. Osteoblast differentiation was induced in confluent cultures by adding 100 nM dexamethasone, 10 mM ${\beta}$-glycerophosphate, 50 mM L-ascorbic acid. The Alizarin red S staining and reverse transcriptase-polymerase chain reaction (RT-PCR) were perfomed to examine the mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OCN), receptor activator for nuclear factor ${\kappa}B$ ligand (RANKL), runt-related transcription factor 2 (RUNX2), collagen-Ⅰ (COL-Ⅰ). There were no significant differences in the osteogenic potentials of DOCs induced from MSCs on ${\beta}-TCP(+/-)$. According to the incubation period, there were significant increasing of Alizadin red S staining in the induction 3 weeks. The mRNA expression of ALP, RUNX2, and RANKL were higher in DOCs/${\beta}-TCP(-)$ than DOCs/${\beta}-TCP(+)$. According to rhBMP-2 concentrations, the mRNA expression of BSP was significantly increased in DOCs/${\beta}-TCP(+)$ compared to that of DOCs/${\beta}-TCP(-)$ on rhBMP 10 ng/ml. Our study presented the ${\beta}-TCP$ will have the possibility that calcium phosphate directly affect the osteoblastic differentiation of the bone marrowderived MSCs.

• Journal of Life Science
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• v.19 no.7
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• pp.845-851
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• 2009

A vector harboring double cassettes; a heterologous gene expression cassette of pHCE-InaN-antigen and a ghost formation cassette of pAPR-cI-E lysis 37 SDM was constructed and introduced to E. coli DH5a. For the production of a bacterial ghost vaccine, bacterial ghosts from E. coli / Streptococcus iniae with four different types of antigens - enolase, GAPDH, sagA and piaA - were produced by the optimization of fermentation parameters such as a glucose concentration of 1 g/l, agitation of 300 rpm and aeration of 1 vvm. Efficiency of ghost bacteria formation was evaluated with cultures of OD$_{600}$=1.0, 2.0 and 3.0. The efficiency of the ghost bacteria formation was 99.54, 99.67, 99.99 and 99.99% with inductions at OD$_{600}$=3.0, 1.0, 2.0 and 1.0 for E. coli/S. iniae antigens enolase, piaA, GAPDH and sagA, respectively. Ghost bacteria as a vaccine was harvested by centrifugation. The antigen protein expressions were analyzed by SDS-PAGE and western blot analysis, and the molecular weights of the enolase, piaA, GAPDH and sagA were 78, 26, 67 and 26 kDa, respectively. The molecular weights of the expressed antigens were consistent with theoretical sizes obtained from the amino acid sequences.

• Molecules and Cells
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• v.41 no.6
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• pp.553-561
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• 2018

Icariside II (ICA II) is used in erectile dysfunction treatment. Adipose tissue-derived stem cells (ADSCs) are efficient at improving erectile function. This study aimed to explore the action mechanism of ADSCs in improving erectile function. ADSCs were isolated from the adipose tissues of rats. Cell proliferation was determined using the Cell Counting Kit-8 (CCK-8) assay. The expressions of mRNA and protein were determined separately through qRT-PCR and western blot. The endogenous expressions of related genes were regulated using recombinant plasmids and cell transfection. A Dual-Luciferase Reporter Assay was performed to determine the interaction between miR-34a and STAT3. Rat models with bilateral cavernous nerve injuries (BCNIs) were used to assess erectile function through the detection of mean arterial pressure (MAP) and intracavernosal pressure (ICP). ICA II promoted ADSCs' proliferation and differentiation to Schwann cells (SCs) through the inhibition of miR-34a. Suppressed miR-34a promoted the differentiation of ADSCs to SCs by upregulating STAT3. ICA II promoted the differentiation of ADSCs to SCs through the miR-34a/STAT3 pathway. The combination of ICA II and ADSCs preserved the erectile function of the BCNI model rats. ADSCs treated with ICA II markedly preserved the erectile function of the BCNI model rats, which was reversed through miR-34a overexpression. ICA II promotes the differentiation of ADSCs to SCs through the miR34a/STAT3 pathway, contributing to erectile function preservation after the occurrence of a cavernous nerve injury.

• Korean Journal of Microbiology
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• v.45 no.4
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• pp.291-299
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• 2009

To elucidate HtrA3's functional roles in the HtrA3 mediated cellular processes, it is necessary to investigate its biochemical characteristics. In the present study, we constructed the plasmids encoding putative mature HtrA3 proteins (M1-HtrA3 and M2-HtrA3) based on the putative maturation sites of highly homologous HtrA1 and mouse HtrA3. We used the pGEX bacterial expression system to develop a simple and rapid purification for the recombinant HtrA3 protein. Although yields of the mature HtrA3 proteins were slightly low as 10~50 ${\mu}g$/L, the amounts and purity of M1- and M2-HtrA3 were enough to investigate their proteolytic activities. The putative mature HtrA3 proteins have proteolytic activity which could cleave $\beta$-casein as an exogenous substrate. We compared the proteolytic activity between the HtrA family, HtrA1, HtrA2, and HtrA3. The cleavage activity of HtrA3 and HtrA2 were 2 folds higher than that of HtrA1, respectively. Our study provides a method for generating useful reagents to identify natural substrates of HtrA3 in the further studies.

• KSBB Journal
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• v.21 no.2
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• pp.133-139
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• 2006

In 'solid-phase' PEGylation, the conjugation reaction occurs as the proteins are attached to a solid matrix, and thus it can have distinct advantages over the conventional, solution-phase process. We report a case study: rhIFN-${\alpha}$-2a was first adsorbed to cation exchange resin and then N-terminally PEGylated by aldehyde mPEG of 5, 10, and 20 kD through reductive alkylation. After the PEGylation, salt gradient elution efficiently recovered the mono-PEGylate in a purified form from the unwanted species such as unmodified IFN, unreacted PEG, and others. The mono-PEGylation and its purification were integrated in a single chromatographic step. Depending on the molecular weight of the mPEG aldehyde used, the mono-PEGylation yield ranged 50-64%. We could overcome the major problems of random, or uncontrollable, multi-PEGylation and the post-PEGylation purification difficulties associated with the solution-phase process. N-terminal sequencing and MALDI-TOF MS confirmed that a PEG molecule was conjugated only to the N-terminus. Compared with the unmodified IFN, the mono-PEGylate showed the reduced anti-viral activity as measured by the cell proliferation assay. The bioactivity was reduced more as the higher molecular weight PEG was conjugated. Immunoreactivity, evaluated indirectly by antibody binding activity using a surface plasmon resonance biosensor, also decreased. Nevertheless, trypsin resistance as well as thermal stability was considerably improved.