• Journal of Microbiology and Biotechnology
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• 제27권4호
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• pp.838-843
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• 2017

Sepsis is a major health problem worldwide, with an extremely high rate of morbidity and mortality, partly due to delayed diagnosis during early disease. Currently, sepsis diagnosis requires bacterial culturing of blood samples over several days, whereas PCR-based molecular diagnosis methods are faster but lack sensitivity. The use of biosensors containing nucleic acid aptamers that bind targets with high affinity and specificity could accelerate sepsis diagnosis. Previously, we used the systematic evolution of ligands by exponential enrichment technique to develop the aptamers Antibac1 and Antibac2, targeting the ubiquitous bacterial peptidoglycan. Here, we show that these aptamers bind to four gram-positive and seven gram-negative bacterial sepsis agents with high binding efficiency. Thus, these aptamers could be used in combination as biological recognition elements in the development of biosensors that are an alternative to rapid bacteria detection, since they could provide culture and amplification-free tests for rapid clinical sepsis diagnosis.

• BMB Reports
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• 제40권2호
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• pp.239-246
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• 2007

Riboflavin synthase from Escherichia coli is a homotrimer of 23.4 kDa subunits and catalyzes the formation of one molecule each of riboflavin and 5-amino-6-ribitylamino- 2,4(1H,3H)-pyrimidinedione by the transfer of a 4-carbon moiety between two molecules of the substrate, 6,7- dimethyl-8-ribityllumazine. Each subunit comprises two closely similar folding domains. Recombinant expression of the N-terminal domain is known to provide a $C_2$-symmetric homodimer. In this study, the binding properties of wild type as well as two mutated proteins of N-terminal domain of riboflavin synthase with various ligands were tested. The replacement of the amino acid residue A43, located in the second shell of riboflavin synthase active center, in the recombinant N-terminal domain dimer reduces the affinity for 6,7-dimethyl-8-ribityllumazine. The mutation of the amino acid residue C48 forming part of activity cavity of the enzyme causes significant $^{19}F$ NMR chemical shift modulation of trifluoromethyl derivatives of 6,7-dimethyl-8-ribityllumazine in complex with the protein, while substitution of A43 results in smaller chemical shift changes.

• BMB Reports
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• 제40권4호
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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.

• Biomolecules & Therapeutics
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• 제13권2호
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• pp.101-106
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• 2005

The generation of secretory IgA antibodies (Abs) for specific immune protection of mucosal surfaces depends on stimulation of the mucosal immune system, but this is not effectively achieved by parenteral or even oral administration of most soluble antigens. Thus, to produce a possible vaccine antigen against urinary tract infections, the uropathogenic E. coli (UPEC) adhesin was genetically coupled to the heat-labile Escherichia coli enterotoxin A2B (ltxa2b) gene and cloned into a pMAL-p2E expression vector. The chimeric construction of pMALfimH/ltxa2b was then transformed into E. coli K-12 TB1 and its nucleotide sequence was verified. The chimeric protein was then purified by applying the affinity chromatography. The purified chimeric protein was confirmed by SDS-PAGE and westem blotting using antibodies to the maltose binding protein (MBP) or the heat labile E. coli subunit B (LTXB), plus the N-terminal amino acid sequence was analyzedd. The orderly-assembled chimeric protein was confirmed by a modified $G_{M1}$-ganglioside ELISA using antibodies to adhesin. The results indicate that the purified chimeric protein was an Adhesin/LTXA2B protein containing UPEC adhesin and the $G_{M1}$-ganglioside binding activity of LTXB. thisstudy also demonstrate that peroral administration of this chimeric immunogen in mice elicited high level of secretory IgA (sIgA) and serum IgG Abs to the UPEC adhesin. The results suggest that the genetically linked LTXA2B acts as a useful mucosal adjuvant, and that adhesin/LTXA2A chimeric protein might be a potential antigen for oral immunization against UPEC.

• 한국재료학회지
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• 제28권6호
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• pp.365-369
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• 2018

We perform density functional theory calculations to study the CO and $O_2$ adsorption chemistry of Pt@X core@shell bimetallic nanoparticles (X = Pd, Rh, Ru, Au, or Ag). To prevent CO-poisoning of Pt nanoparticles, we introduce a Pt@X core-shell nanoparticle model that is composed of exposed surface sites of Pt and facets of X alloying element. We find that Pt@Pd, Pt@Rh, Pt@Ru, and Pt@Ag nanoparticles spatially bind CO and $O_2$, separately, on Pt and X, respectively. Particularly, Pt@Ag nanoparticles show the most well-balanced CO and $O_2$ binding energy values, which are required for facile CO oxidation. On the other hand, the $O_2$ binding energies of Pt@Pd, Pt@Ru, and Pt@Rh nanoparticles are too strong to catalyze further CO oxidation because of the strong oxygen affinity of Pd, Ru, and Rh. The Au shell of Pt@Au nanoparticles preferentially bond CO rather than $O_2$. From a catalysis design perspective, we believe that Pt@Ag is a better-performing Pt-based CO-tolerant CO oxidation catalyst.

• 대한의생명과학회지
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• 제16권3호
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• pp.201-206
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• 2010

In order to develop procedures for the rapid isolation of recombinant sugar transporter in functional form from away from the endogenous insect cell transporter, gene fusion techniques were exploited. Briefly, BamH1-digested human HepG2 type glucose transport protein cDNA was first cloned into a transfer vector pBlueBacHis, containing a tract of six histidine residues. Recombinant baculoviruses including the human cDNA were then generated by allelic exchange following transfection of insect cells with wild-type BaculoGold virus DNA and the recombinant transfer vector. Plaque assay was then performed to obtain and purify recombinant viruses expressing the human transport protein. All the cell samples that had been infected with viruses from the several blue plaques exhibited a positive reaction in the immnuassay, demonstrating expression of the glucose transport protein. In contrast, no color development in the immunoassay was observed for cells infected with the wild-type virus or no virus. Immunoblot analysis showed that a major immunoreactive band of apparent Mr 43,000~44,000 was evident in the lysate from cells infected with the recombinant baculovirus. Following expression of the recombinant fusion protein with the metal-binding domain and enterokinase cleavage site, the fusion protein was recovered by competition with imidizole using immobilized metal charged resin. The leader peptide was then removed from the fusion protein by cleavage with porcine enterokinase. Final separation of the recombinant protein of the interest was achieved by passage over $Ni^{2+}$-charged resin under binding conditions. The expressed transport protein bound cytochalasin B and demonstrated a functional similarity to its human counterpart.

• 한국생물정보학회:학술대회논문집
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• 한국생물정보시스템생물학회 2003년도 제2차 연례학술대회 발표논문집
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• pp.277-287
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• 2003

Acetolactate synthase (ALS, EC 4.1.3.18; also referred to as acetohydroxy acid synthase) catalyzes the first common step in the biosynthesis of valine, leucine, and isoleucine in microorganisms and plants. Recently X-ray structure of yeast ALS was available. Pair-wise alignment of yeast and tobacco ALS sequences revealed 63% sequence similarity. Using Deep View and automatic modeling on Swiss model server, we have generated reliable models of tobacco ALS based on yeast ALS template with a calculated pair-wise RMSD of 0.86 Angstrom. Functional roles of four residues located on the subunit interface (H142, El43, M350, and R376) were examined by site-directed mutagenesis. Seven mutants were generated and purified, of which three mutants (H142T, M350V, and R376F) were found to be inactivated under various assay conditions. The H142k mutant showed moderately altered kinetic properties. The E143A mutant increased 10-fold in K$_m$ value while other parameters remained unchanged. The M350C mutant was strongly resistant to three tested herbicides, while the R376k mutant can bind with herbicide carder at similar affinity to that of wild type enzyme, as determined by tryptophan quenching study. Except M350V mutant, all other mutants were ate to bind with cofactor FAD. Taken together, it is likely that residues H142 and E143 are located at the active site, while residues M350 and R376 are possibly located at the overlapping region of active site and herbicide binding site of the enzyme. Our data also allows us to hypothesize that the interaction between side chains of residues M350 and R376 are probably essential for the correct conformation of the active site. It remains to be elucidated that, whether the herbicide, upon binding with enzyme, inactivates the enzyme by causing change in the active site allosterically, which is unfavorable for catalytic activity.

• BMB Reports
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• 제28권5호
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• pp.437-442
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• 1995

The potency of yeast-derived methionyl-free human growth hormone (rhGH), which was obtained by removal of the N-terminal Met from methionyl-hGH, was estimated by in vitro and in vivo assays. In radio-receptor assay where the binding affinity of growth hormone to the receptor was estimated, the recombinant hGH showed 2.9 international units (IU) per mg of specific activity. In contrast, pitUitary-derived human growth hormone had a slightly lower receptor binding activity (2.5 IU/mg) compared with recombinant growth hormone. For the in vivo assay, efficacy of rhGH was tested by use of hypophysectomized rats, in which pituitary organs were surgically removed, resulting in the termination of growth hormone secretion. The weight-increase in rats by the injection of rhGH was almost identical to the result obtained by the injection of the same amount of pituitary-derived (international standard) hGH. A comparision of the secondary structures of rhGH and rMet-hGH by circular dichroism spectrophotometer demonstrated that the removal of the methionyl residue from rMet-hGH did not exert any effect on the structure of the growth hormone. In conclusion, methionyl-free human growth hormone produced from yeast was highly potent in biological activity and maintained a legitimate three dimensional structure.

• BMB Reports
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• 제30권6호
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• pp.397-402
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• 1997

A thiol-specific spin label was attached to cysteine-102 of yeast cytochrome c and electron paramagnetic resonance (EPR) spectra were measured as a function of added cytochrome c oxidase concentration. The intensity decreased due to line broadening as cytochrome c formed a complex with cytochrome c oxidase and reached a minimum when the ratio of cytochrome c to cytochrome c oxidase became one. Replacement of either Lys-72 or Lys-87 of cytochrome c by Glu did not result in a significant change in binding affinity. Interestingly the K72E mutant, unlike K87E, had a much lower rate of electron transfer than the wild type. These results indicate that many positively charged residues as a group participate in complex formation but Lys-72 might be important for cytochrome c to be locked in an orientation for an efficient electron transfer. A stoichiometry of 1 was also confirmed by optical absorption of the cytochrome c-cytochrome c oxidase complex which had been run through a gel chromatography cloumn to remove unbound cytochrome c. The EPR spectrum of this 1:1 complex, however, was a mixture of two components. This explains a biphasic kinetics for a single binding site on cytochrome c oxidase without invoking conformational transition.

• Biomolecules & Therapeutics
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• 제11권3호
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• pp.157-162
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• 2003

The generation of secretory IgA antibodies(Abs) for specific immune protection of mucosal surfaces depends on stimulation of the mucosal immune system, but this is not effectively achieved by parenteral or even oral administration of most soluble antigens. Thus, to produce a possible vaccine antigen against urinary tract infections, the uropathogenic E. coli (UPEC) adhesin was genetically coupled to the ctxa2b gene and cloned into a pMAL-p2E expression vector. The chimeric construction of pMALfimHIctxa2b was then transformed into E. coli K-12 TB1 and its nucleotide sequence was verified. The chimeric protein was then purified by applying the affinity chromatography. The purified chimeric protein was confirmed by SDS-PAGE and western blotting using antibodies to the maltose binding protein (MBP) or the cholera toxin subunit B (CTXB), plus the N-terminal amino acid sequence was analyzed. The orderly-assembled chimeric protein was confirmed by a modified $G_{M1}$-ganglioside ELISA using antibodies to adhesin. The results indicate that the purified chimeric protein was an Adhesin/CTXA2B protein containing UPEC adhesin and the $G_{M1}$-ganglioside binding activity of CTXB. This study also demonstrate that peroral administration of this chimeric immunogen in mice elicited high level of secretory IgA and serum IgG Abs to the UPEC adhesin. The results suggest that the genetically linked CTXA2B acts as a useful mucosal adjuvant, and that the adhesin/CTXA2B chimeric protein might be a potential antigen for oral immunization against UPEC.