• Asian-Australasian Journal of Animal Sciences
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• v.29 no.1
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• pp.126-133
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• 2016

A gene from Actinomyces sp. Korean native goat (KNG) 40 that encodes an endo-${\beta}$-1,4-glucanase, EG1, was cloned and expressed in Escherichia coli (E. coli) $DH5{\alpha}$. Recombinant plasmid DNA from a positive clone with a 3.2 kb insert hydrolyzing carboxyl methyl-cellulose (CMC) was designated as pDS3. The entire nucleotide sequence was determined, and an open-reading frame (ORF) was deduced. The ORF encodes a polypeptide of 684 amino acids. The recombinant EG1 produced in E. coli $DH5{\alpha}$ harboring pDS3 was purified in one step using affinity chromatography on crystalline cellulose and characterized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis/zymogram analysis of the purified enzyme revealed two protein bands of 57.1 and 54.1 kDa. The amino terminal sequences of these two bands matched those of the deduced ones, starting from residue 166 and 208, respectively. Putative signal sequences, a Shine.Dalgarno-type ribosomal binding site, and promoter sequences related to the consensus sequences were deduced. EG1 has a typical tripartite structure of cellulase, a catalytic domain, a serine-rich linker region, and a cellulose-binding domain. The optimal temperature for the activity of the purified enzyme was $55^{\circ}C$, but it retained over 90% of maximum activity in a broad temperature range ($40^{\circ}C$ to $60^{\circ}C$). The optimal pH for the enzyme activity was 6.0. Kinetic parameters, $K_m$ and $V_{max}$ of rEG1 were 0.39% CMC and 143 U/mg, respectively.

• Proceedings of the Microbiological Society of Korea Conference
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• 1991.04a
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• pp.225-236
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• 1991

The genes encoding the thermostable $\alpha$-amylase and maltogenic amylase from Bacillus lichenciformis were cloned and expressed in E. coli. The recombinant plasmid pTA322 was found to contain a 3.1kb EcoRI genomic DNA fragment of the thermostable $\alpha$-amylase. The cloned $\alpha$-amylase was compared with the B. licheniformis native $\alpha$-amylase. Both $\alpha$-amylase have the same optimal temperature of $70^{\circ}C$ and are stable in the pH range of 6 and 9. The complete nucleotide sequences of the thermostable $\alpha$-amylase gene were determined. It was composed of one open reading rame of 1,536 bp. Start and stop codons are ATG and TAG. From the amino acid sequence deduced from the nucleotide sequence, the cloned thermostable $\alpha$-amylase is composed of 483 amino acid residues and its molecular weight is 55,200 daltons. The content of guanine and cytosine is $47.46mol\%$ and that of third base codon was $53_41mol\%$. The recombinant plasmid, pIJ322 encoding the maltogenic amylase contains a 3.5kb EcoRI-BamHI genomic DNA fragment. The optimal reaction temperature and pH of the maltogenci amylase were $50^{\circ}C$ and 7, respectively. The maltogenic amylase was capable of hydrolysing pullulan, starch and cyclodextrin to produce maltose from starch and panose from pullulan. The maltogenic amylase also showed the transferring activity. The maltogenic amylase gene is composed of one open reading frame of 1,734bp. Start and stop codons are ATG and ATG. At 2bp upstream from start codon, the nucleotide sequence AAAGGGGGAA seems to be the ribosome-binding site(RBS, Shine-Dalgarno sequence). A putative promoter(-35 and-10 regions) was found to be GTTAACA and TGATAAT. From deduced amino acid sequence from the nucleotide srquence, this enzyme was comosed of 578 amino acid residues and its molecular weight was 77,233 daltons. The content of guanine and cytosine was $48.1mol\%$. The new recombinant plasmid, pTMA322 constructed by inserting the thermostable $\alpha$-amylase gene in the EcoRI site of pIJ322 to produce both the thermostable $\alpha$-amylase and the maltogenic amylase were expressed in the E. coli. The two enzymes expressed from E. coli containing pTMA322 was reacted with the $15\%$ starch slurry at $40^{\circ}C$ for 24hours. The distribution of the branched oligosaccharides produced by the single-step process was of the ratio 50 : 50 between small oligosaccharide up DP3 and large oligosaccharide above DP3.

• Journal of Life Science
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• v.19 no.6
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• pp.809-817
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• 2009

The phenotypes associated with a gene function are often the best clue to its role in the plant. Transgenic plants ectopically expressing or suppressing a gene can provide useful information related to the gene function. In this study, we constructed three vectors - pFGL571, pFGL846 and pFGL847 - for the Agrobacterium-mediated ectopic expression of plant genes using pPZP211 and modified CaMV 35S, UBQ3 or UBQ10 promoters. The three vectors have several merits such as small size, high copy in bacteria, enough restriction enzyme sites in multi cloning sites and nucleotide sequence information. Analysis of transgenic plants containing GUS or sGFP reporter genes under the control of modified CaMV 35S, UBQ3 or UBQI0 promoter revealed that all of the three promoters showed high activities during most developmental stages after germination and in floral organs. Furthermore, we generated a RNAi module vector, pFGL727, to suppress plant gene expressions and confirmed that pFGL727 is useful for the suppression of a gene expression using rice transgenic plants. Taken together, our new vectors would be very useful for the ectopic expression or the suppression of plant genes.

• Korean Journal of Microbiology
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• v.40 no.4
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• pp.348-354
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• 2004

The expression and antibacterial. activity of recombinant human lactoferrin (hLf) was studied from meth­ylotrophic yeast Pichia pastoris. The gene encoding hLf, isolated from human breast cDNA library, was subcloned into the expression vector, pPIC3.5K under the control of AOX1 promoter. The gene was integrated into the host chromosome and was identified by Southern blotting. The expression of the integrated gene was investigated by RT-PCR, Northern blotting, SDS-PAGE and Western blotting. Discrete band corresponding to hLf was detected from the SDS-PAGE, which was confirmed by Western blotting. The expression was also confirmed by RT-PCR and Northern blotting. The antibacterial activity of the recombinant hLf (rhLf) was investigated using Staphy­lococcus aureus ATCC 6538P and Micrococcus flavus ATCC 10240 as test organisms. The rhLf showed strong antibacterial activities against the bacteria. Furthermore, many Gram-negative animal pathogens such as E.coli ATCC8739, 25922, and Salmonella typhimurium 114 and 115, Pseudomonas fluorescens ID 963 I, P. aeruginosa KCCM 11802, and Gram-positive bacteria Bacillus mesentericus were also inhibited in their growth by the rhLf.

• Korean Journal of Microbiology
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• v.43 no.3
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• pp.166-172
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• 2007

ERM proteins transfer the methyl group to $A_{2058}$ in 23S rRNA, which reduces the affinity of MLS (macrolide-lincosamide-streptogramin B) antibiotics to 23S rRNA, thereby confer the antibiotic resistance on micro-organisms ranging from antibiotic producers to pathogens and are classified into monomethyltransferase and dimethyltransferase. To investigate the differences between mono- and dimethyltransferase, tirD, a representative monomethylase gene was cloned in Escherichia coli from Streptomyces fradiae which contains ermSF, dimethylase gene as well to overexpress the TlrD for the first time. T7 promoter driven expression system successfully overexpress tlrD as a insoluble aggregate at $37^{\circ}C$ accumulating to around 55% of the total cell protein but unlike ErmSF, culturing at temperature as low as $18^{\circ}C$ did not make insoluble aggregate of protein into soluble protein. Coexpression of Thioredoxin and GroESL, chaperone was not helpful in turning into soluble protein either as in case of ErmSF. These results might suggest that differences between mono- and dimethylase could be investigated on the basis of the characteristics of protein structure. However, a very small amount of soluble protein which could not be detected by SDS-PAGE conferred antibiotic resistance on E. coli as in ErmSF which was expected from the activity exerted by monmethylase in a cell.

• Korean Journal of Microbiology
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• v.41 no.1
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• pp.1-7
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• 2005

Methylophaga aminosulfidovorans SK1 (KCTC 10323 BP) can utilize trimethylamine as a sole carbon, nitrogen, and energy source. The bacterial flavin-containing monooxygenase (bFMO) gene was identified in the strain and the recombinant enzyme expressed in E. coli oxidized trimethylamine. To study the function and regulation of the bfmo, over 8,000 nucleotide sequences of the neighboring regions including the bfmo were determined. Three open reading frames proceeding to the bfmo gene encoded analogues to highly conserved nitrate/nitrite sensing two-component system regulators and a methyl accepting protein. Two small open reading frames just downstream of the bfmo gene showed no similar proteins of known functions but the sequences were conserved among other bacteria. Reverse transcription-polymerase chain reaction analysis showed that the six putative genes consisted of three transcription units. The three regulatory genes located upstream of the bfmo gene formed two separate transcription units. The bfmo and the two downstream genes were transcribed from a single promoter.

• Microbiology and Biotechnology Letters
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• v.41 no.1
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• pp.17-25
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• 2013

The thermotolerant methylotrophic yeast Hansenula polymorpha is an attractive model organism for various fundamental studies, such as the genetic control of enzymes involved in methanol metabolism, peroxisome biogenesis, nitrate assimilation, and resistance to heavy metals and oxidative stresses. In addition, H. polymorpha has been highlighted as a promising recombinant protein expression host, especially due to the availability of strong and tightly regulatable promoters. In this study, we investigated the possibility of employing human serum albumin (HSA) as the fusion tag for the secretory expression of heterologous proteins in H. polymorpha. A set of four expression cassettes, which contained the methanol oxidase (MOX) promoter, translational HSA fusion tag, and the terminator of MOX, were constructed. The expression cassettes were also designed to contain sequences for accessory elements including His8-tag, $2{\times}(Gly_4Ser_1)$ linkers, tobacco etch virus protease recognition sites (Tev), multi-cloning sites, and strep-tags. To determine the effects of the size of the HSA fusion tag on the secretory expression of the target protein, each cassette contained the HSA gene fragment truncated at a specific position based on its domain structure. By using the Green fluorescence protein gene as the reporter, the properties of each expression cassette were compared in various conditions. Our results suggest that the translational HSA fusion tag is an efficient tool for the secretory expression of recombinant proteins in H. polymorpha.

• Journal of Plant Biotechnology
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• v.41 no.2
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• pp.81-88
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• 2014

Salt cress (Thellungiella halophila or Thellungiella parvula), species closely related to Arabidopsis thaliana, represents an extremophile adapted to harsh saline environments. To isolate salt-tolerance genes from this species, we constructed a cDNA library from roots and leaves of salt cress plants treated with 200 mM NaCl. This cDNA library was subsequently shuttled into the destination binary vector [driven by the cauliflower mosaic virus (CaMV) 35S promoter] designed for plant transformation and expression via recombination- assisted cloning. In total, 305,400 pools of transgenic BASTA-resistant lines were generated in Arabidopsis using either T. halophila or T. parvula cDNA libraries. These were used for functional screening of genes involved in salt tolerance. Among these pools, 168,500 pools were used for primary screening to date from which 7,157 lines showed apparent salt tolerant-phenotypes in the initial screen. A secondary screen has now identified 165 salt tolerant transgenic lines using 1,551 (10.6%) lines that emerged in the first screen. The prevalent phenotype in these lines includes accelerated seed germination often accompanied by faster root growth compared to WT Arabidopsis under salt stress condition. In addition, other lines showed non-typical development of stems and flowers compared to WT Arabidopsis. Based on the close relationship of the tolerant species to the target species we suggest this approach as an appropriate method for the large-scale identification of salt tolerance genes from salt cress.