• Korean Journal of Veterinary Research
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• v.37 no.3
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• pp.541-546
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• 1997

There are two conserved regions with a significantly high amino acid sequence homology among the A subunits of STX, SLTs and ricin. To produce an inactive Verotoxin-2 (VT-2), two different mutants, pE167D and pDE5A, were constructed by site-directed mutagenesis, respectively, on the basis of the previous reports that two regions lie within the active-site clefts of the A subunits of ricin and STX family. The cytotoxicity ($10^3$ $CD_{50}/ml$) of VT-2 holotoxin with E167D mutation was reduced by $10^3$-fold compared with wild-type level. In addition, VT-2 with DE5A ($Trp_{202}GlyArgIleSer_{206}$) deletion mutation showed a significantly low cytotoxicity ($10^1$ $CD_{50}/ml$), resulting in $10^5$- and $10^2$-fold reductions, respectively, compared with the wild-type and E167D mutatant. SDS-PAGE for protein samples showed a 33-kDa band corresponding to the A subunit of VT-2. These results indicate that reduction in cytotoxic activity was affected not by amount of VT-2 protein produced but by mutation.

• Journal of Life Science
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• v.24 no.12
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• pp.1269-1275
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• 2014

TALEN is a newly developed gene engineering method to knock out specific genes. It contains a DNA binding domain and a Fok1 nuclease domain in the TALEN plasmid. Therefore, the engineered TALEN construct can bind to any region of genomic DNA and cut the target nucleotide, thereby inducing mutation. In this study, we constructed two TALEN constructs targeted to a protein initiation codon (DBEX2) or the 25th upstream region (DBPR25) to enable mRNA synthesis of SETDB1 HMTase. We performed the TALEN cloning in two steps. The first step was from module vectors to pFUS array vectors. We confirmed successful cloning with a colony PCR experiment and Esp31 restriction enzyme digestion, which resulted in a smear band and a 1 Kb insert band, respectively The second step of the cloning was from a pFUS array vector to a mammalian TALEN expression vector. The engineered TALEN construct was sequenced with specific primers in an expression vector. As expected, a specific array from the module vectors was shown in the sequencing analysis. The specific module sequences were regularly arrayed in every 100 bp, and SETDB1 expression totally disappeared in the TALEN-DBEX2 transfection. PCR amplification targeting of DBEX2 was performed, and the PCR product was digested with a T7E1 restriction enzyme. The expression of SETDB1 was down-regulated in the TALEN-DBPR25 transfection. Morphological changes were also observed in the two TALEN constructs with transfected HeLa cells. These results suggest that the engineered TALEN constructs in two strategic approaches are very useful to knock-out of the SETDB1 gene and to study gene function.

• Korean Journal of Microbiology
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• v.31 no.4
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• pp.279-285
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• 1993

Alterations of the p53 gene arc among the most frequent genetic changes in human cancer and often result in increased levels of p53 protein within the malignant cells. Detection of accumulated p53 protein can be a useful prognostic tool in human cancer. In order to make polyclonal antibodies for immunohistochemical screening. human p53 gene was expressed in E. coli in the form of GST (glutathione S-transfi.:rase) fusion proteins. Two p53 gene fragments. which were N('()I small fragment encoding amino acid residues of 1-151-: and Ncol large fragment of 159-393. were subeloned into the unique BamHI site present within the pGEX-2T vector using BamHI linker and recombinant plasmids pGTNS and pGTNL were constructed. respectively. The p53 cDNA fragment (from pC53-$SN_3$,) encoding amino acid 38-145 (proline at residue 72) was amplified by polymerase chain reaction(PCR). The amplified DNA was digested with BamHI and Prull and inserted into the BamHI-Smal sites of pG EX-2T and recombinant plasmid pGTBP was constructed. After IPTG induction of these plasmids for 4 hours. fusion proteins were purified from E. coli extracts with glutathione Sepharose beads. The bound proteins were resolved by 10% SDS-polyacrylamide gel electrophoresis and the molecular weights were 54 kDa. 53 kDa and 40 kDa. respectively. Approximately one milligram of fusion proteins were purified from 1 -liter cultures.

• Korean Journal of Microbiology
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• v.45 no.3
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• pp.233-238
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• 2009

The expression of Corynebacterium ammoniagenes purF was analyzed by utilizing a plasmid carrying a cat gene fused to the purF promoter region. Adenine and guanine repressed the expression of the purF gene by 20~30% but hypoxanthine did not exert such repressive effect. The expression purF was maximal at the late log phase and remained constant throughout the stationary phase. Promoter $P_{180}$ which was developed in C. glutamicum was also functional in C. ammoniagenes, achieving maximal activity at the late log phase. The promoter outperformed Escherichia coli $P_{tac}$ promoter by 40~50% level. DNA-affinity purification identified a protein which could bind to the promoter region of the purF gene. The protein showed high similarity to the CRP-family transcriptional regulator encoded by NCgl0120 in C. glutamicum. The size of the screened protein agreed with the expected protein size from the ORF NCgl0120. The corresponding gene in C. ammoniagenes encoded a 42 kDa polypeptide composed of 400 amino acids with expected pI of 4.9. The encoded protein showed 14.1% and 15.8% identity with E. coli and Bacillus subtilis PurR, respectively, suggesting that the isolated protein might be a novel type of regulatory protein involved in the regulation of purine metabolism.

• Korean Journal of Microbiology
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• v.44 no.2
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• pp.93-97
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• 2008

RraA is a recently discovered protein inhibitor that regulates the enzymatic activity of RNase E, which plays a major role in the decay and processing of RNAs in Escherichia coli. It has also been shown to regulate the activity of RNase ES, a functional Streptomyces coelicolor ortholog of RNase E, which has 36% identity to the amino-terminal region of RNase E. There are two open reading frames in S. coelicolor genome that can potentially encode proteins having more than 35.4% similarity to the amino acid sequence of RraA. DNA fragment encoding one of these RraA orthologs, designated as RraAS2 here, was amplified and cloned in to E. coli vector to test whether it has ability to regulate RNase E activity in E. coli cells. Co-expression of RraAS2 partially rescued E. coli cells over-producing RNase E from growth arrest, although not as efficiently as RraA, induced by the increased ribonucleolytic activity in the cells. The copy number of ColEl-type plasmid in these cells was also decreased by 14% compared to that in cells over-producing RNase E only, indicating the ability of RraAS2 to inhibit RNase E action on RNA I. We observed that the expression level of RraAS2 was lower than that of RraA by 4.2 folds under the same culture condition, suggesting that because of inefficient expression of RraAS2 in E. coli cells, co-expression of RraAS2 was not efficiently able to inhibit RNase E activity to the level for proper processing and decay of all RNA species that is required to restore normal cellular growth to the cells over-producing RNase E.

• Applied Microscopy
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• v.41 no.4
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• pp.249-255
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• 2011

Neurons utilize a large quantity of energy for their survival and function, and thereby require active mitochondrial function. Mitochondrial morphology shows dynamic changes, depending on the cellular condition, and mitochondrial dynamics are required for neuronal development and function. In this study, we found that the length of mitochondria in the distal axon is significantly shorter than that of mitochondria in dendrites or proximal axons of cerebral cortical neurons, and the reason for this difference is the local fission within the axon. We also found that suppression of Drp1, a key regulator of mitochondrial fission, resulted in significant elongation of mitochondria in axons. Collectively, these results suggest that local mitochondrial fission within the axon contributes to region-dependent mitochondrial length differences in the axons of cortical neurons.

• 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.

• Journal of Life Science
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• v.25 no.5
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• pp.507-514
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• 2015

D-Xylulose is phosphorylated to D-xylulose-5-phosphate by D-xylulose kinase before it enters glycolysis via the nonoxidative pentose phosphate pathway. A gene encoding a novel D-xylulose kinase (XK) from K. gwangalliensis strain SJ2 was sequenced and expressed in E. coli. The sequence of the isolated XK gene was 1,419 bp, encoding 472 amino acids. The XK protein was more closely related to the Arthrobacter phenanthrenivorans XK than to the Bifidobacterium catenulatum one, as reflected in the sequence identity (54.9% vs. 38.7%). The XK gene was subcloned into the pCold-II expression vector. The resulting plasmid was transformed into E. coli strain BL21 (DE3) cells and the expression of the recombinant XK protein was induced by the addition of IPTG. The resulting protein was expressed as a fusion protein of approximately 48 kDa containing a N-terminal six-histidine extension that was derived from the expression vector. The expressed protein was homogenized by affinity chromatography and showed enzymatic activity corresponding to D-xylulose kinase. XK enzyme kinetic studies with D-xylulose and ATP showed a Km of 250±20 μM and 1,300±50 μM, respectively. The results obtained from this study will provide a wider knowledge base for the characterization of D-xylulose kinase at the molecular level.

• Journal of Life Science
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• v.20 no.6
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• pp.934-939
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• 2010

We have previously reported recombinant productions of bacteriocins using yeast expression plasmid pAUR123, which contains the alcohol dehydrogenase (ADH) promoter, in Saccharomyces cerevisiae cells and their antibacterial activities. In order to improve the antibacterial activities of bacteriocidal yeast cells, a strong glyceraldehyde phosphate dehydrogenase (GPD) promoter gene of S. cerevisiae was amplified and inserted upstream into bacteriocin genes such as the OR-7, Subpeptin JM4-A or JM4-B gene in the corresponding recombinant yeast plasmid. Yeast cells transformed by the recombinant plasmid containing the GPD promoter represented higher antibacterial activities against both Gram positive B. subtilis and Gram negative E. coli cells compared to those transformed by the corresponding recombinant plasmid containing the ADH promoter. Thus, yeast cells harboring the recombinant plasmid containing the GPD promoter constructed in this study could be applied in the food preservative or animal feed industries.

• Journal of Life Science
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• v.24 no.1
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• pp.1-7
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• 2014

Eukaryotic gene expression is an important process, which is initiated by several transcription factors and RNA polymerases that occupy the promoter region of genomic DNA. Although there are many experiments to identify the promoter region in a gene, it is time and labor consuming to finalize it. In this study, we utilized bioinformatic programs, including Ensembl, NCBI, and CpG plots, to identify the cloning promoter region in SETDB1 genomic DNA. We performed PCR amplification to obtain the SETDB1 promoter on an approximately 2 kb region upstream from the TSS named SETDB1-P1. The PCR product was ligated with TA cloning vectors, and we confirmed the insert size using restriction enzyme digestion. Sequentially, the insert was subcloned into a pGL3-luc vector to produce pGL3-SETDB1- P1-luc and then confirmed by DNA sequencing. We also obtained a fragmented PCR product called P2 and P3 and performed a luciferase assay using pGL3-SETDB1-P1-luc transfection. We found that several anticancer drugs, including taxol, 4-FU, and doxorubicin, decreased the promoter activity of SETDB1. We obtained consistent data on the regulation of SETDB1 gene expression after anticancer drug treatment using Western blot analysis and RT-PCR. Our results suggest that promoter cloning of the human SETDB1 gene utilizing bioinformatics is a very useful and timesaving approach to study gene expression.