• Journal of Biomedical Engineering Research
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• v.44 no.6
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• pp.465-475
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• 2023

In this study, we demonstrated a silica nanofibrous membrane based on the electrospinning process and evaluated its DNA isolation and purification performance in PCR pretreatment. Generally, silica membranes made of non-woven fabric are used for PCR pretreatment, but this study aimed to improve the efficiency of the pretreatment process by developing a nanofiber-type silica membrane with high specific surface area and porosity. In order to manufacture a nanofiber-shaped silica film while maintaining the original physical properties of silica, nanofiber membranes produced by adding various concentrations of PEO (5 wt%, 8 wt%, and 10 wt%) to silica prepared by the sol-gel method were compared. In terms of nanofiber membrane production, the higher the PEO concentration, the more effective it was in producing nanofiber membranes. The produced silica nanofiber membrane was inserted to a pretreatment device used in commercial PCR equipment, and the pretreatment performance was compared and verified using Salmonella bacteria. When Salmonella was used, samples containing 5 wt% PEO showed superior PCR efficiency compared to samples containing 8 wt% and 10 wt% PEO. These results show that adding 5 wt% of PEO can effectively improve DNA purification and separation by producing a nanofiber-shaped silica film while maintaining the physical properties of silica. We expect that this study will contribute to the development of effective PCR pretreatment technology essential for various molecular biology applications.

• Journal of Life Science
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• v.15 no.6 s.73
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• pp.851-856
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• 2005

Although valuable microbes have been isolated from the soil for the various productions of useful components, the microbes which can be cultivated in the laboratory are only $0.1-1\%$ of all microbes. To solve this problem, the study has recently been tried for making the valuable components from the environment by directly separating unculturable micrbial DNA in the soil. But it is known that humic acid originated from the soil interrupts various restriction enzymes and molecular biological process. Thus, in order to prevent these problems, this study modified the method separated soil DNA with phenol, CTAB and PEG. In order to compare the degree of purity for each DNA and the molecular biological application process, $A_{260}/A_{280}$ ratio, restriction enzymes, and PCR were performed. In case of DNA by the modified method, total yield of DNA was lower but $A_{260}/A_{280}$ ratio was higher than the previously reported methods. It was confirmed that the degree of purity is improved by the modified method. But it was not cut off by all kinds of tested restriction enzymes because of the operation of a very small amount of interrupting substances. When PCR was operated with each diluted DNA in different concentrations and GAPDH primer, the DNA by the modified method could be processed for PCR in the concentration of 100 times higher than by the previously reported separation method. Therefore, this experiment can find out the possibility of utilization for the unknown substances by effectively removing the harmful materials including humic acid and help establishing metagenomic DNA library from the soil DNA having the high degree of purity.

• Journal of Microbiology and Biotechnology
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• v.17 no.7
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• pp.1090-1097
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• 2007

Genomic analysis of Thermococcus sp. NA1 revealed the presence of a 3,927-base-pair (bp) family B-type DNA polymerase gene, TNA1_pol. TNA1_pol, without its intein, was overexpressed in Escherichia coli, purified using metal affinity chromatography, and characterized. TNA1_pol activity was optimal at pH 7.5 and $75^{\circ}C$. TNA1_pol was highly thermostable, with a half-life of 3.5h at $100^{\circ}C$ and 12.5h at $95^{\circ}C$. Polymerase chain reaction parameters of TNA1_pol such as error-rate, processivity, and extension rate were measured in comparison with rTaq, Pfu, and KOD DNA polymerases. TNA1_pol averaged one incorrect bp every 4.45 kilobases (kb), and had a processivity of 150 nucleotides (nt) and an extension rate of 60 bases/s. Thus, TNA1_pol has a much faster elongation rate than Pfu DNA polymerase with 7-fold higher fidelity than that of rTaq.

• Animal cells and systems
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• v.2 no.4
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• pp.539-543
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• 1998

Hrp1, of Schizosaccharomyces pombe, is a new member of the SW12/SNF2 protein family that contains a chromodomain and a DNA binding domain as well as ATPase/7 helicase domains. This configuration suggests that Hrp1 could be a homolog of mouse CHD1, which is thought to function in altering the chromatin structure to facilitate gene expression. To understand the enzymatic nature of Hrp1 we purified the 6-Histidine-tagged Hrp1 protein (6$\times$His-Hrp1) to homogeneity from a S. pombe Hrp1-overexpressing strain and hen examined its biochemical properties. We demonstrate that the purified 6$\times$His-Hrp1 protein exhibited a DNA-binding activity with a moderate preference to the (A＋T)-rich tract in double-stranded NA via a minor groove interaction. However, we failed to detect any intrinsic DNA helicase activity from the purified Hrp1 like other SW12/SNF2 proteins. These observations suggest that the DNA binding activities of Hrp1 may be involved in the remodeling of the chromatin structure with DNA-dependent ATPase. We propose that Hrp1 may function in heterochromatins as other proteins with a chromo- or ATPase/helicase domain and play an important role in the determination of chromatin architecture.

• Korean Journal of Environmental Agriculture
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• v.30 no.4
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• pp.367-376
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• 2011

BACKGROUND: In order to develop effective assessment method for Korean paddy soil microbial community structure, reliable genomic DNA extraction method from paddy soil and quantitative real-time PCR (qRT-PCR) method are needed to establish METHODS AND RESULTS: Out of six conventional soil genomic DNA extraction methods, anion exchange resin purification method was turn to be the most reliable. Various PCR primers for distinguishing five bacterial phylum (${\alpha}$-Proteobacteria, ${\beta}$-Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes), all bacteria, and all fungi were tested. Various qRT-PCR temperature conditions were also tested by repeating experiment. Finally, both genomic DNA extraction and qRT-PCR methods for paddy soil were well established. CONCLUSION: Quantitative real-time PCR (qRT-PCR) method to assess paddy soil microbial community was established.

• Journal of Microbiology
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• v.45 no.6
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• pp.593-596
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• 2007

In this study, we describe the development of a simple and efficient method for cell lysis via the insertion of a bacteriophage lambda lysis gene cluster into the pET22b expression vector in the following order; the T7 promoter, a gene for a target protein intended for production, Sam7 and R. This insertion of R and Sam7 into pET22b exerted no detrimental effects on cellular growth or the production of a target protein. The induction of the T7 promoter did not in itself result in the autolysis of cells in culture but the harvested cells were readily broken by freezing and thawing. We compared the efficiency of the cell lysis technique by freezing and thawing to that observed with sonication, and determined that both methods completely disintegrated the cells and released proteins into the solution. With our modification of pET22b, the lysis of cells became quite simple, efficient, and reliable. This strategy may prove useful for a broad variety of applications, particularly in experiments requiring extensive cell breakage, including library screening and culture condition exploration, in addition to protein purification.

• Journal of Life Science
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• v.11 no.4
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• pp.385-391
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• 2001

Filamentous hemagglutinin (FHA) is considered as an essential immunogenic component for incorporation into acellular vaccines against Bordetella pertussis, the causative agent of whooping cough. Classically, antipertussis vaccination has employed an intramuscular route. An alternative approach to stimulate mucosal and systemic immune responses is oral immunization with recombinant live vaccine carrier strains of Salmonella typhimurium. An attenuated live Salmonella vaccine sgrain($\Delta$cya $\Delta$crp) expressing recombinant FHA(rFHA) was developed. Stable expressionof rFHA was achieved by the use of balanced-lethal vector-host system. which employs an asd deletion in the host chromosome to impose in obligate requirement for diaminopimelic acid. The chromosomal $\Delta$asd mutation was complemented by a plasmid vector possessing the asd$^{+}$ gene. A 3 kb DNA fragment encoding immuno dominant regionof FHA was subcloned in-frame downstream to the ATG translation initiation codon in the multicopy Asd$^{+}$ pYA3341 vector to create pYA3457. Salmonella vaccine harboring pYA3457 expressed approximately 105kDa rFHA protein. The 100% maintenance of [YA3457 in vaccine strain was confirmed by stability examinations. Additionally, a recombinant plasmid pYA3458 was constructed to overpress His(8X)-tagged rFHA in Essherichia coli. His-tagged rFHA was purified from the E. coli strain harboring pYA3458 using Ni$^{2+}$-NTA affinity purification system.>$^{2+}$-NTA affinity purification system.

• BMB Reports
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• v.43 no.8
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• pp.541-546
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• 2010

We utilized a mammalian expression system to purify and characterize autotaxin (ATX)/lysophospholipase D, an enzyme present in the blood responsible for biosynthesis of lysophosphatidic acid. The human ATX cDNA encoding amino acids 29-915 was cloned downstream of a secretion signal of CD5. At the carboxyl terminus was a thrombin cleavage site followed by the constant domain (Fc) of IgG to facilitate protein purification. The ATX-Fc fusion protein was expressed in HEK293 cells and isolated from conditioned medium of a stable clone by affinity chromatography with Protein A sepharose followed by cleavage with thrombin. The untagged ATX protein was further purified to essential homogeneity by gel filtration chromatography with a yield of approximately 5 mg/liter medium. The purified ATX protein was enzymatically active and biologically functional, offering a useful tool for further biological and structural studies of this important enzyme.

• Journal of the Korean Magnetic Resonance Society
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• v.19 no.2
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• pp.95-98
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• 2015

Reverse gyrase is a hyperthermophile specific protein which introduces positive supercoils into DNA molecules. Reverse gyrase consists of an N-terminal helicase-like domain and a C-terminal topoisomerase domain. The helicase-like domain shares the three-dimensional structure with two tandem RecA-folds (H1 and H2), in which the subdomain H2 is interrupted by the latch domain (H3). To understand the physical property of the hyperthermophile-specific protein, two subdomains af_H1 and af_H23 have been cloned into E. coli expression vector, pET28a. The $^{15}N$-labeled af_H1 and af_H23 proteins were expressed and purified for heteronuclear NMR study. The af_H1 protein exhibits the well-dispersion of amide signals in its $^1H/^{15}N$-HSQC spectra and thus further NMR study continues to be progressed.

• Journal of the Korean Magnetic Resonance Society
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• v.14 no.2
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• pp.105-116
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• 2010

SH3YL1, a novel protein containing one Src homology 3 domain at the carboxyl terminus was first detected in mouse anagen skin cDNA. This protein had a significant homology with YHRO 16c/Ysc 84, the yeast Src homology 3 domain-containing protein. The sequence identity was remarkable at the carboxyl and amino-terminal Src homology 3 domain, suggesting that the novel protein is a mouse homolog of the yeast protein and thus was termed as SH3YL1. SH3YL1 is composed of two domains, a DUF500 at N-termini and a SH3 domain at C-termini. In our study we cloned the SH3 domain in bacterial expression system in Escherichia coli using pET32a vector with TEV protease cleavage site and purified as a monomer using affinity chromatography. The N-terminal poly-Histidine tag was cleaved with TEV protease and target protein was used for backbone studies. Our study showed that SH3 domain primarily consists of $\beta$-sheet which is in consistence with previous result performed on the truncated SH3 domain of SH3YL1.