• Journal of Microbiology and Biotechnology
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• 제18권7호
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• pp.1245-1251
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• 2008

Oxalate decarboxylases (OXDCs) (E.C. 4.1.1.2) are enzymes catalyzing the conversion of oxalate to formate and $CO_2$. The OXDCs found in fungi and bacteria belong to a functionally diverse protein superfamily known as the cupins. Fungi-originated OXDCs are secretory enzymes. However, most bacterial OXDCs are localized in the cytosol, and may be involved in energy metabolism. In Agrobacterium tumefaciens C58, a locus for a putative oxalate decarboxylase is present. In the study reported here, an enzyme was overexpressed in Escherichia coli and showed oxalate decarboxylase activity. Computational analysis revealed the A. tumefaciens C58 OXDC contains a signal peptide mediating translocation of the enzyme into the periplasm that was supported by expression of signal-peptideless and full-length versions of the enzyme in A. tumefaciens C58. Further site-directed mutagenesis experiment demonstrated that the A. tumefaciens C58 OXDC is most likely translocated by a twin-arginine translocation (TAT) system.

• BMB Reports
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• 제44권10호
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• pp.692-697
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• 2011

To investigate the pathways of oxidoreductases in plants, 2 key components in thioredox systems i.e. thioredoxin h (Trx h) and NADPH-dependent thioredoxin reductase (NTR) genes were first isolated from tomatoes (Solanum lycopersicum). Subsequently, the coding sequences of Trx h and NTR were inserted into pET expression vectors, and overexpressed in Escherichia coli. In the UV-Visible spectra of the purified proteins, tomato Trx h was shown to have a characteristic 'shoulder' at ~290 nm, while the NTR protein had the 3 typical peaks unique to flavoenzymes. The activities of both proteins were demonstrated by following insulin reduction, as well as DTNB reduction. Moreover, both NADPH and NADH could serve as substrates in the NTR reduction system, but the catalytic efficiency of NTR with NADPH was 2500-fold higher than with NADH. Additionally, our results reveal that the tomato Trx system might be involved in oxidative stress, but not in cold damage.

• Journal of Microbiology and Biotechnology
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• 제22권4호
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• pp.510-515
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• 2012

A recombinant putative dextransucrase (DexT) was produced from Leuconostoc citreum KM20 as a 160 kDa protein, but its productivity was very low (264 U/l). For optimization, we examined enzyme activity in 7 Escherichia coli strains with inducer molecules such as lactose or IPTG. E. coli BL21-CodonPlus(DE3)-RIL exhibited the highest enzyme activity with lactose. Finally, DexT activity was remarkably increased by 12-fold under the optimized culture conditions of a cell density to start induction ($OD_{600}$) of 0.95, a lactose concentration of 7.5 mM, and an induction temperature of $17^{\circ}C$. These results may effectively apply to the heterologous expression of other large DexT genes.

• Journal of Microbiology and Biotechnology
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• 제17권11호
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• pp.1868-1874
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• 2007

We have developed a robotic system for an automated parallel cell cultivation process that enables screening of induction parameters for the soluble expression of recombinant protein. The system is designed for parallelized and simultaneous cultivation of up to 24 different types of cells or a single type of cell at 24 different conditions. Twenty-four culture vessels of about 200 ml are arranged in four columns${\times}$six rows. The system is equipped with four independent thermostated waterbaths, each of which accommodates six culture vessels. A two-channel liquid handler is attached in order to distribute medium from the reservoir to the culture vessels, to transfer seed or other reagents, and to take an aliquot from the growing cells. Cells in each vessel are agitated and aerated by sparging filtered air. We tested the system by growing Escherichia coli BL21(DE3) cells harboring a plasmid for a model protein, and used it in optimizing protein expression conditions by varying the induction temperature and the inducer concentration. The results revealed the usefulness of our custom-made cell cultivation robot in screening optimal conditions for the expression of soluble proteins.

• Journal of Microbiology and Biotechnology
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• 제19권3호
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• pp.257-264
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• 2009

A $\beta$-agarase gene, agaB34, was functionally cloned from the genomic DNA of a marine bacterium, Agarivorans albus YKW-34. The open reading frame of agaB34 consisted of 1,362 bp encoding 453 amino acids. The deduced amino acid sequence, consisting of a typical N-terminal signal peptide followed by a catalytic domain of glycoside hydrolase family 16 (GH-16) and a carbohydrate-binding module (CBM), showed 37-86% identity to those of agarases belonging to family GH-16. The recombinant enzyme (rAgaB34) with a molecular mass of 49 kDa was produced extracellularly using Escherichia coli $DH5{\alpha}$ as a host. The purified rAgaB34 was a $\beta$-agarase yielding neoagarotetraose (NA4) as the main product. It acted on neoagarohexaose to produce NA4 and neoagarobiose, but it could not further degrade NA4. The maximal activity of rAgaB34 was observed at $30^{\circ}C$ and pH 7.0. It was stable over pH 5.0-9.0 and at temperatures up to $50^{\circ}C$. Its specific activity and $k_{cat}/K_m$ value for agarose were 242 U/mg and $1.7{\times}10^6/sM$, respectively. The activity of rAgaB34 was not affected by metal ions commonly existing in seawater. It was resistant to chelating reagents (EDTA, EGTA), reducing reagents (DTT, $\beta$-mercaptoethanol), and denaturing reagents (SDS and urea). The E. coli cell harboring the pUC18-derived agarase expression vector was able to efficiently excrete agarase into the culture medium. Hence, this expression system might be used to express secretory proteins.

• Biotechnology and Bioprocess Engineering:BBE
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• 제10권3호
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• pp.225-229
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• 2005

To investigate impediments to plasmid transformation in Brevibacterium flavum BF4 and B. lactofermentum BL1, cell surface barriers were determined by measuring growth inhibition whilst enzymatic barriers were determined by comparing DNA methylation properties. B. lactofermentum was more sensitive to growth inhibition by glycine than B. flavum. Release of cellular proteins during sonication was more rapid for B. lactofermentum than for B. flavum. Plasmid DNA (pCSL 17) isolated from B. flavum transformed recipient $McrBC^+$ strains of Escherichia coli with lower efficiency than $McrBC^-$. McrBC digestion of this DNA confirmed that B. flavum contain methylated cytidines in the target sequence of McrBc sequences but B. lactofermentum contained a different methylation pattern. DNA derived from the B. lactofermentum transformed recipient $EcoKR^+$ strains of E. coli with lower efficiency than $EcoKR^-$, indicating the presence of methylated adenosines in the target sequence of EcoK sequences. The present data describe the differences in the physical and enzymatic barriers between two species of corynebacteria and also provide some insight into the successful foreign gene expression in corynebacteria.

• Journal of Microbiology and Biotechnology
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• 제5권1호
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• pp.6-13
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• 1995

A gene coding for the $\beta$-galactosidase (lactase) of Kluyveromyces tragilis UCD 55-55 was isolated by complementation in Escherichia coli YMC9. From the plasmid library made from Sau3A-digested chromosomal DNA, one positive clone was selected. The cloned gene for $\beta$-galactosidase was on 7.3 kilobase pair DNA fragment, and a slightly low level of $\beta$-galactosidase enzyme activity was detecied in E. coli. It was also confirmed that the cloned gene comes from K. tragilis by DNA-DNA hybridization and immunochemical blotting experiments. In order to construct a new yeast strain having the metabolic ability for lactose, the cloned gene for K. tragilis $\beta$-galactosidase was inserted in yeast vector YEp24 and YRp17, and transformed into Saccharomyces cerevisiae YNN27 and Ml-2B. The yeast transformants showed the nearly the same $\beta$-galactosidase productivity as level of K. tragilis when uninduced, but these could not utilize lactose as a sole carbon source, presumably due to the lack of lactose transport system. Nevertheless, a slightly higher ethanol productivity was achieved by these transformants than S. cerevisiae or K. tragilis, in the medium containing glucose and lactose.

• 대한수의학회지
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• 제53권4호
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• pp.217-224
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• 2013

Transgenic plants have been tested as an alternative host for the production and delivery of experimental oral vaccines. Here, we developed transgenic potatoes that express the major antigenic sites A and D of the glycoprotein S from transmissible gastroenteritis coronavirus (TGEV-$S_{0.7}$) under three expression vector systems. The DNA integration and mRNA expression level of the TGEV-$S_{0.7}$ gene were confirmed in transgenic plants by PCR and northern blot analysis. Antigen protein expression in transgenic potato was determined by western blot analysis. Enzyme-linked immunosorbent assay results revealed that based on a dilution series of Escherichia coli-derived antigen, the transgenic line P-2 had TGEV-$S_{0.7}$ protein at levels that were 0.015% of total soluble proteins. We then examined the immunogenicity of potato-derived TGEV-$S_{0.7}$ antigen in mice. Compared with the wild-type potato treated group and synthetic antigen treated group, mice treated with the potato-derived antigen showed significantly higher levels of immunoglobulin (Ig) G and IgA responses.

• Journal of Microbiology and Biotechnology
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• 제19권12호
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• pp.1557-1564
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• 2009

Human CDX2 is known as a caudal-related homeodomain transcription factor that is expressed in the intestinal epithelium and is important in differentiation and maintenance of the intestinal epithelial cells. The caudal-related homeobox proteins bind DNA according to a helix-turn-helix structure, thereby increasing the structural stability of DNA. A cancer-tumor suppressor role for Cdx2 has been shown by a decrease in the level of the expression of Cdx2 in colorectal cancer, but the mechanism of transcriptional regulation has not been examined at the molecular level. We developed a large-scale system for expression of the recombinant, novel CDX2, in Escherichia coli. A highly purified and soluble CDX2 protein was obtained in E. coli strain BL21(DE3)RIL and a hexahistidine fusion system using Ni-NTA affinity column, anion exchange, and gel filtration chromatographies. The identity and secondary structure of the purified CDX2 protein were confirmed by MALDI-TOF MS, Western blot, and a circular dichroism analyses. In addition, we studied the DNA-binding activity of recombinant CDX2 by ELISA experiment and isolated human CDX2-binding proteins derived from rat cells by an immobilized GST-fusion method. Three CDX2-binding proteins were found in the gastric tissue, and those proteins were identified to the homeobox protein Hox-D8, LIM homeobox protein 6, and SMC1L1 protein.

• Journal of Microbiology
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• 제45권2호
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• pp.175-178
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• 2007

An endonuclease from Xanthomonas oryzae pathovar oryzae KACC 10331, XorII, was recombinantly produced in Escherichia coli using a T7 system. XorII was purified using a combination of ion exchange and immobilized metal affinity chromatography (IMAC). An optimized washing protocol was carried out on an IMAC in order to obtain a high purity product. The final amount of purified XorII was approximately 2.5 mg/L of LB medium. The purified recombinant XorII was functional and showed the same cleavage pattern as PvuI. The enzyme activity tested the highest at $25^{\circ}C$ in 50 mM NaCl, 10 mM Tris-HCl, 10 mM $MgCl_{2}$, and 1 mM dithiothreitol at a pH of 7.9.