• 환경생물
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• 제22권3호
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• pp.472-477
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• 2004

본 연구에서는 감마선으로 유도된 돌연변이체들에서 공통으로 발현되는 방사선 관련 유전자들의 발현을 연구하기 위하여, B. lentimorbus WJ5 의 방사선 유도 돌연변이체에서 발현되는 유전자를 DNA microarray로 동시에 탐색하였다. DNA microarray는 B. lentimorbus WJ5 genome을 무작위로 절단하여 2,000 단편으로 구성하였으며, 감마선 $(^{60}/Co)$으로 유도된 7 돌연변이체의 발현을 정량적으로 관찰하였다. 클러스터 분석결과 발현된 408 유전자 중 27개가 감마선 유도 돌연변이체 모두에서 유의하게 발현이 증가되었다. 특히, 복구(mutL, mutM) 에너지 대사 (acsA, sdhB, pgk, yhjB, citB), protease (npr), 산화자극에 대한 환원 (HMM)관련 유전자들이 동시에 증가되었다. 이는 감마선 유도 돌연변이체들에서 자발적인 직/간접 복구 관련 유전자의 발현 증가는 방사선 노출 직후 보이는 stress response와는 다른 현상임을 나타내는 것으로 생각된다.

• International Journal of Industrial Entomology and Biomaterials
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• 제9권2호
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• pp.217-223
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• 2004

A cathepsin L-like cysteine protease, v-cath, encoded by the baculovirus has been shown to playa role in host liquefaction. We have identified a v-cath gene in the silkworm virus, Bombyx mori nuclear polyhedrosis virus (BmNPV) K1 strain. The 969 bp v-cath has an open reading frame of 323 amino acids. A putative cleavage site and catalytic sites were conserved in BmNPV-K1 v-cath. The predicted three-dimensional structure of BmNPV-K1 v-cath revealed that the overall fold of BmNPV-K1 v-cath is similar to that of other proteases of the papain family. The deduced amino acid sequence of BmNPV-K1 v-cath showed 98% and 97% protein sequence identity to BmNPV T3 strain and to Autographa californica nuclear polyhedrosis virus, respectively. The BmNPV-K1 v-cath differed at 4 amino acid positions from BmNPV T3. The v-cath gene in BmNPV-K1 genome is located on the EcoRV 6 kb and XhoI 9 kb fragments. Northern hybridization analysis of BmNPV K1 v-cath gene revealed that it is expressed late in infection.

• 미생물학회지
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• 제30권1호
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• pp.60-64
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• 1992

The secretion vector with promoter and signal sequence region of neutral protease gene (npr) from Bacillus amyloliquefaciens was constructed by the technique of polymerase chain reaction (PCR). A unique restriction iste was introduced into the 3' of the signal coding region by the synthesis of PCR primer. To demonstrate the function of cloned promoter and signal sequence, we used the E. coli .betha.-lactamase structural gene as a foreign gene. The signal sequence of .betha.-lactamase gene was deleted by Bal31 exonuclease and only mature region was introduced into the secretion vector. Bacillus subtilis cells transformed by the recombinant vector synthesized the fusion protein and were also capable of removing the signal peptide from the original fusion protein, as judged by the assay of .betha.-lactamase activity and secretion into the growth medium by western blotting.

• Parasites, Hosts and Diseases
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• 제43권3호
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• pp.101-110
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• 2005

In this study, the trypsin gene (bgtryp-1) from the German cockroach, Blattella germanica, was cloned via the immunoscreening of patients with allergies to cockroaches. Nucleotide sequence analysis predicted an 863 bp open reading frame which encodes for 257 amino acids. The deduced amino acid sequence exhibited $42-57\%$ homology with the serine protease from dust mites, and consisted of a conserved catalytic domain (GOSGGPLV). bgtryp-1 was determined by both Northern and Southern analysis to be a 0.9 kb, single-copy gene. SDS-PAGE and Western blotting analyses of the recombinant protein (Bgtryp-1) over-expressed in Escherichia coli revealed that the molecular mass of the expressed protein was 35 kDa, and the expressed protein was capable of reacting with the sera of cock-roach allergy patients. We also discussed the possibility that trypsin excreted by the digestive system of the German cockroach not only functions as an allergen, but also may perform a vital role in the activation of PAR-2.

• 대한한의학회지
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• 제21권3호
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• pp.174-185
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• 2000

Objectives : This study was carried out to evaluate the effects of five fractions on cell viability, cell cycle progression and apoptosis. Methods : This study employed MTT assay, Cell cycle analysis, Cpp32 protease assay, DNA fragmentation assay and Quantitative RT-PCR analysis. Results : In MTT assay, the butanol fraction of Injinsaryung-San has showed magnificent viability, while the $H_2O$ fraction and ethylacetate fraction also showed higher viability than the control group. The $H_2O$ fraction of Injinsaryung-San has showed magnificent viability, and butanol fraction and ethylacetate fraction of Injinsaryung-San with etoposide have also showed higher viability than the only etoposide group. Cell cycle analysis showed that each fraction of Injinsaryung-San had no significant effect on the cell cycle. DNA fragmentation assay showed that the butanol fraction, $H_2O$ fraction and ethylacetate fraction carried inhibitory effects on apoptosis induction. Cpp32 protease activity assay showed that the butanol fraction, $H_2O$ fraction and ethylacetate fraction decreased Cpp32 protease activity, with the butanol fraction displaying greater effects. Quantitative RT-PCR showed that the butanol fraction, $H_2O$ fraction and ethylacetate fraction suppressed Fas and Bax genes, the butanol fraction increased BcI-2 gene, however no effect on Cpp32. Conclusions : The data shows that the butanol fraction of Injinsaryung-San increases the hepatocyte viability and has the heptocelluar protective effect by the suppression of apoptosis through gene regulation.

• Journal of Microbiology and Biotechnology
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• 제11권2호
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• pp.222-228
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• 2001

Vibrio metschnikovii strain RH530 is a non-pathogenic, industrially-important alkaline protease producer which has been isolated from wastewater. In this paper, we report on the transformation of this strain by using the method of electroporation. A field strength of $7.5\;kVcm^{-1}$ and $25\;{\mu}F$, and using a 0.2-cm cuvette, appeared to be the optimal conditions for electroporation of the cells with the recombinant pSBCm plasmid carrying the vapK alkaline protease gene and the ColE1 replicon. Cells were subjected to osmotic shock in order to remove extracelluar DNase, and adding 200 mM of sucrose to electroporation buffer cells showed an increased transformation efficiency. Maximum efficiency of transformation was obtained at an early exponential growth phase. Using all of the conditions mentioned above, we routinely obtained a transformation efficiency of more than $10^4{({\mu}g\;plasmid\;DNA)}^{-1}$. The stability of the plasmid pSBCm in V. metschnikovii RH530 was 25% after 18h of growth (27 generations) in the medium without antibiotic selection. The insertion of the par locus to the pSBCm increased the stability of the plasmid up to 42% without selective pressure. The increase in plasmid stability was accompanied by the increase in the productivity of alkaline protease in the recombinant V. metschnikovii strain RH530. Determining optimal conditions for the transformation of the industrially-important, nonpathogenic Vibrio strain, and the improvement of plasmid stability by introducing the par locus into the high copy number plasmid vector, will allow the development of procedures involved in the genetic manipulation of this strain, particularly for its use in the production of industrial enzymes such as alkaline protease.

• 한국수산과학회지
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• 제47권4호
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• pp.370-375
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• 2014

BACE2 is a membrane-bound aspartic protease that is highly homologous with BACE1. While BACE1 processes the amyloid precursor protein (APP) at a key step in generating ${\beta}$-amyloid peptide and presumably causes Alzheimer's disease (AD), BACE2 has not been demonstrated to be involved in APP processing directly, and its physiological functions are unknown. To determine its function and to develop inhibitors from marine sources, we constructed an overexpression vector for producing BACE2. The gene encoding human BACE2 protease was amplified using the polymerase chain reaction and cloned into the pET11a expression vector, resulting in pET11a/BACE2. Recombinant BACE2 protease was overexpressed successfully in E. coli as inclusion bodies, refolded using the rapid-dilution method, and purified via two-step fast protein liquid chromatography using Sephacryl S-300 gel filtration and Resource-Q column chromatography. The BACE2 protease produced was an active form. This study provides an efficient method not only for studying the basic properties of BACE2, but also for developing inhibitors from natural marine sources.

• 생명과학회지
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• 제17권2호통권82호
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• pp.272-278
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• 2007

단백질분해효소는 다른 단백질들의 아미노산 간에 존재하는 peptide 결합을 절단하며 생리학적, 상업적 측면에서 중요한 위치를 차지하는 효소의 한 부류이다. 이러한 단백질분해효소의 새로운 공급원을 찾기 위하여 비교적 저온에서 체외단백질분해효소를 생산하는 세균을 동해심층수로부터 분리하였다. 분리된 균 중 저온에서의 생육정도와 높은 활성을 가지는 균주를 선별하여 HJ 47이라 명명하였다. 형태학적, 생리생화학적 특성과 16s rRNA gene의 염기서열을 조사한 후 Pseudoalteromonas sp.에 포함하는 것으로 나타났다. 분리된 Pseudoalteromonas sp. HJ 47은 $10^{\circ}C$에서도 비교적 잘 자랐으며, $37^{\circ}C$에서 최적의 생육을 보여 주었다. 최적생육온도와는 달리 배양시간당 최대 체외단백질분해효소의 생산은 $20^{\circ}C$에서 최대였고 대수기 후반과 정지기에 생산이 시작되어 15시간 경과 후 최대의 생산을 보여주었다. 효소활성의 최적온도는 $35^{\circ}C$, 최적 pH는 8로 판명되었다.

• BMB Reports
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• 제35권5호
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• pp.498-507
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• 2002

A gene that encodes a thermostable protease, coined thermicin, has been isolated from Thermoanaerobacter yonseiensis that is expressed and characterized in E. coli.. In order to elucidate the molecular characteristics on thermostability of the enzyme, molecular modeling and mutagenesis technology were applied. In the modeling structure, the structural core, including the active site, was well conserved; whereas, the two loop regions were unique when compared to thermitase. The mutant enzyme with the small loop deleted (D190-I196), based on modeling structural information, showed identical enzyme activity. However, when the large loop was deleted (P233-P244), a little lower $K_m$ and even a lower kcat was found. This indicates that the large loop could influence catalytic activity. However, the unfolding temperature ($T_m$), which was determined by a differential-scanning calorimetry for the mutant enzyme deleted the small loop, was $96^{\circ}C$. This is $14^{\circ}C$ lower than that for the parent thermicin. These results suggest that the small loop may play a role in maintaining the proper folding of the enzyme at high temperatures, whereas the large loop might be related to catalysis.

• 한국수산과학회지
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• 제42권5호
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• pp.461-467
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• 2009

A bacterial strain was screened and identified from sea sediments in Tongyeong coastal area in order to obtain proteases or peptidase cleaving C-terminal of glutamic acid. Strain TS0611, which showed the highest activity from 5 isolated protease producing strains, was selected and its properties investigated. Strain TS0611 was a gram-positive rod, $1.2\;{\mu}m$ in cell length, catalase positive, motility-positive, melanin-negative and grew at 15~$50^{\circ}C$, and hydrolyzed gelatin and casein. This strain was identified as Bacillus thuringiensis or Bacillus cereus based on results from the API system(API 50 CHB) which examined saccharides properties, fatty acid composition of cell wall, and 16S rRNA gene sequence.