• Food Science of Animal Resources
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• v.37 no.1
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• pp.134-138
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• 2017

Salmonella enterica infects a broad range of host animals, and zoonostic infection threatens both public health and the livestock and meat processing industries. Many antimicrobials have been developed to target Salmonella envelope that performs essential bacterial functions; however, there are very few analytical methods that can be used to validate the efficacy of these antimicrobials. In this study, to develop a potential biosensor for Salmonella envelope stress, we examined the transcription of the S. enterica serovar typhimurium spy gene, the ortholog of which in Escherichia coli encodes Spy (${\underline{s}}pheroplast$ ${\underline{p}}rotein$ ${\underline{y}}$). Spy is a chaperone protein expressed and localized in the periplasm of E. coli during spheroplast formation, or by exposure to protein denaturing conditions. spy expression in S. typhimurium was examined by constructing a spy-gfp transcriptional fusion. S. typhimurium spy transcription was strongly induced during spheroplast formation, and also when exposed to membrane-disrupting agents, including ethanol and the antimicrobial peptide polymyxin B. Moreover, spy induction required the activity of regulator proteins BaeR and CpxR, which are part of the major envelope stress response systems BaeS/BaeR and CpxA/CpxR, respectively. Results suggest that monitoring spy transcription may be useful to determine whether a molecule particularly cause envelope stress in Salmonella.

• Genomics & Informatics
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• v.7 no.2
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• pp.53-56
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• 2009

Recent evidence has strongly suggested that the CAP/TC10 pathway is involved in the trafficking, docking, and fusion of vesicles containing the insulin-responsive glucose transporter Glut4 to the plasma membrane. However, little is known about how the genes employed in the CAP/TC10 pathway are associated with the development of type 2 diabetes mellitus. In this study, we sequenced 4 genes of the CAP/TC10 pathway [SORBS1, CBL, CRK, and RHOQ] in 24 individuals to identify genetic variations in these loci. A total of 48 sequence variants were identified, including 23 novel variations. To investigate the possible association with type 2 diabetes mellitus, 3 single nucleotide polymorphisms from SORBS1, 3 from CBL, and 4 from RHOQ were genotyped in 1122 Korean type 2 diabetic patients and 1138 nondiabetic controls. Using logistic regression analysis, 1 significant association between SNP rs1376405 in RHOQ and type 2 diabetes mellitus [OR = 8.714 (C.I. 1.714-44.29), p = 0.009] was found in the recessive model. Our data demonstrate a positive association of the RHOQ gene in the CAP/TC10 pathway with T2DM in the Korean population.

• Journal of Microbiology
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• v.44 no.3
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• pp.293-300
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• 2006

The Escherichia coli heat-labile enterotoxin B subunit (HLT-B) is one of the most powerful mucosal immunogens and known mucosal adjuvants. However, the induction of high levels of HLT-B expression in E. coli has proven a difficult proposition. Therefore, in this study, the HLT-B gene was cloned from pathogenic E. coli and expressed as a fusion protein with GST (glutathion S-transferase) in E. coli BL2l (DE3), in an attempt to harvest a large quantity of soluble HLT-B. The culture conditions, including the culture media used, temperature, pH and the presence of lactose as an inducer, were all optimized in order to obtain an increase in the expression of soluble GST-rHLT-B. The biological activity of the purified rHLT-B was assayed in a series of GMI-ELISA experiments. The findings of these trials indicated that the yield of soluble recombinant GST-rHLT-B could be increased by up to 3-fold, as compared with that seen prior to the optimization, and that lactose was a more efficient alternative inducer than IPTG. The production of rHLT-B, at 92 % purity, reached an optimal level of 96 mg/l in a 3.7 L fermentor. The specific GM1 binding ability of the purified rHLT-B was determined to be almost identical to that of standard CTB.

• Journal of Microbiology and Biotechnology
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• v.20 no.12
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• pp.1689-1695
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• 2010

Pantothenate kinase (PanK) catalyzes the first step in the biosynthesis of the essential and ubiquitous cofactor coenzyme A (CoA) in all organisms. Here, we report the identification, cloning, and characterization of panK-sp from Streptomyces peucetius ATCC 27952. The gene encoded a protein of 332 amino acids with a calculated molecular mass of 36.8 kDa and high homology with PanK from S. avermitilis and S. coelicolor A3(2). To elucidate the putative function of PanK-sp, it was cloned into pET32a(+) to construct pPKSP32, and the PanK-sp was then expressed in E. coli BL21(DE3) as a His-tag fusion protein and purified by immobilized metal affinity chromatography. The enzyme assay of PanK-sp was carried out as a coupling assay. The gradual decrease in NADH concentration with time clearly indicated the phosphorylating activity of PanK-sp. Furthermore, the ca. 1.4-fold increase of DXR and the ca. 1.5-fold increase of actinorhodin by in vivo overexpression of panK-sp, constructed in pIBR25 under the control of a strong $ermE^*$ promoter, established its positive role in secondary metabolite production from S. peucetius and S. coelicolor, respectively.

• BMB Reports
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• v.41 no.12
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• pp.868-874
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• 2008

Neurofilaments (NFs) are neuronal intermediate filaments composed of light (NF-L), middle (NF-M), and heavy (NF-H) subunits. NF-L self-assembles into a "core" filament with which NF-M or NF-H co-assembles to form the neuronal intermediate filament. Recent reports show that point mutations of the NF-L gene result in Charcot-Marie-Tooth disease (CMT). However, the most recently described rod domain mutant of human NF-L (A148V) has not been characterized in cellular level. We cloned human NF-L and used it to engineer the A148V. In phenotypic analysis using SW13 cells, A148V mutation completely abolished filament formation despite of presence of NF-M. Moreover, A148V mutation reduced the levels of in vitro self-assembly using GST-NF-L (H/R) fusion protein whereas control (A296T) mutant did not affect the filament formation. These results suggest that alanine at position 148 is essentially required for NF-L self-assembly leading to subsequent filament formation in neuronal cells.

• Journal of Microbiology and Biotechnology
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• v.18 no.7
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• pp.1235-1244
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• 2008

Indole-3-acetic acid (IAA) is produced commonly by plants and many bacteria, however, little is known about the genetic basis involving the key enzymes of IAA biosynthetic pathways from Bacillus spp. IAA intermediates from the Gram-positive spore-forming bacterium Paenibacillus polymyxa E681 were investigated, which showed the existence of only an indole-3-pyruvic acid (IPA) pathway for IAA biosynthesis from the bacterium. Four open reading frames (ORFs) encoding indole-3-pyruvate decarboxylase-like proteins and putative indole-3-pyruvate decarboxylase (IPDC), a key enzyme in the IPA synthetic pathway, were found on the genome sequence database of P. polymyxa and cloned in Escherichia coli DH5$\alpha$. One of the ORFs, PP2_01257, was assigned as probable indole-3-pyruvate decarboxylase. The ORF consisted of 1,743 nucleotides encoding 581 amino acids with a deduced molecular mass of 63,380 Da. Alignment studies of the deduced amino acid sequence of the ORF with known IPDC sequences revealed conservation of several amino acids in PP2_01257, essential for substrate and cofactor binding. Recombinant protein, gene product of the ORF PP2_01257 from P. polymyxa E681, was expressed in E. coli BL21 (DE3) as a glutathione S-transferase (GST)-fusion protein and purified to homogeneity using affinity chromatography. The molecular mass of the purified enzyme showed about 63 kDa, corresponding closely to the expected molecular mass of IPDC. The indole-3-pyruvate decarboxylase activity of the recombinant protein, detected by HPLC, using IPA substrate in the enzyme reaction confirmed the identity and functionality of the enzyme IPDC from the E681 strain.

• Journal of Microbiology and Biotechnology
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• v.23 no.1
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• pp.56-63
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• 2013

We have characterized the putative ${\alpha}$-glucosidase gene (st2525) selected by total genome analysis from the acidothermophilic crenarchaeon Sulfolobus tokodaii strain 7. The ORF was cloned and expressed as a fusion protein in Escherichia coli, and recombinant ST2525 was purified by Ni-NTA affinity chromatography. Maximum activity was observed at $95^{\circ}C$ and pH 4.0, and the enzyme exhibited stability with half-lives of 40.1 min and 7.75 min at extremely high temperatures of $100^{\circ}C$ and $105^{\circ}C$, respectively. The enzyme retained at least 85% of its maximal activity in the pH range of 4.0-11.0. ST2525 exclusively hydrolyzed ${\alpha}$-1,4-glycosidic linkages of oligosaccharides in an exo-type manner, with highest catalytic efficiency toward maltotriose. The enzyme also displayed transglycosylation activity, converting maltose to isomaltose, panose, maltotriose, isomaltotriose, etc. From these results, ST2525 could be potentially useful for starch hydrolysis as well as novel synthesis of oligosaccharides in industry.

• Parasites, Hosts and Diseases
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• v.39 no.3
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• pp.241-246
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• 2001

The antigenic domain of the major surface protein (Nc-p43) of Neospora caninum was examined by polymerase chain reaction of its gene fragments and recombinant expression as GST fusion proteins. The fragments of Nc-p43 were as follow: a total open reading frame (OFR), T: OFR without signal sequence and C-terminal hydrophobic sequence, S: N-terminal 2/3 parts of S, A: C-terminal 2/3 parts, P; N-terminal 1/3 part, X: middle 1/3 part Y; and C-terminal 1/3 part, Z, respectively. The DNA fragments were cloned into pGEX-47 vector. Recombinant plasmids transformed into Escherichia coli of BL21 pLysS (DE3) strain were induced to express GST or GST fused fragments of Nc-p43 such as 69 kDa protein for T,66 kDa for S, 52 kDa for A,53 kDa for P, and 40 kDa proteins for X, Y, and Z, respectively in SDS-PAGE. The Nc-p43 fragments of T, S, and P reacted with a bovine serum of neosporosis while those of A, X, Y, and Z together with GST did not in the western blot. These findings suggest that the antigenic domain of Nc-p43 of N. caninum may be localized in the C-terminal 2/3 parts. Together with Al9 clone in SAGI of Toxoplasma gondii (Nam et at., 1996), the P fragment of Nc-p43 could be used as efficient antigens to diagnose and differentiate those infections with both species .

• Parasites, Hosts and Diseases
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• v.45 no.2 s.142
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• pp.87-94
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• 2007

In this study, we describe Korean isolates of Trichomonas vaginalis infected with double-stranded (ds) RNA virus (TVV). One T. vaginalis isolate infected with TVV IH-2 evidenced weak pathogenicity in the mouse assay coupled with the persistent presence of a dsRNA, thereby indicating a hypovirulence effect of dsRNA in T. vaginalis. Cloning and sequence analysis results revealed that the genomic dsRNA of TVV IH-2 was 4,647 bp in length and evidenced a sequence identity of 80% with the previously-described TVV 1-1 and 1-5, but only a 42% identity with TVV 2-1 and 3 isolates. It harbored 2 overlapping open reading frames of the putative capsid protein and dsRNA-dependent RNA polymerase (RdRp). As previously observed in the TVV isolates 1-1 and 1-5, a conserved ribosomal slip-page heptamer (CCUUUUU) and its surrounding sequence context within the consensus 14-nt overlap implied the gene expression of a capsid protein-RdRp fusion protein, occurring as the result of a potential ribosomal frameshift event. The phylogenetic analysis of RdRp showed that the Korean TVV If-2 isolate formed a compact group with TVV 1-1 and 1-5 isolates, which was divergent from TVV 2-1, 3 and other viral isolates classified as members of the Giardiavirus genus.

• Molecules and Cells
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• v.23 no.3
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• pp.316-322
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• 2007

Zinc finger transcription factor genes are a significant fraction of the genes in the vertebrate genome. Here we report the isolation and characterization of a human zinc finger-containing gene, ZNF435, from a fetal brain cDNA library. ZNF435 cDNA is 1290 base pairs in length and contains an open reading frame encoding 349 amino acids with four C2H2-type zinc fingers at its carboxyl terminus and a SCAN motif at its amino terminus. RT-PCR results showed that ZNF435 was expressed in all tested tissues. A ZNF435-GFP fusion protein was located in the nucleus and the four zinc fingers acted as nuclear localization signals (NLSs). ZNF435 was found to be capable of homo-association, and this effect was independent of its zinc fingers. Furthermore, ZNF435 proved to be a transcription repressor as its overexpression in AD293 cells inhibited the transcriptional activities of AP-1.