• 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.

• Applied Biological Chemistry
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• 제38권1호
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• pp.7-12
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• 1995

본 연구는 lacZ' 유전자의 promoter 개발을 위하여 착수하였다. lacZ' 유전자의 heterologous promoter I과 II를 효모 염색체의 Bam HI DNA 단편에서 분리하였다. Promoter I의 크기는 2.5 Kb 정도이고 ${\beta}-galactosidase$ 활성은 124.6 U/mg protein이었으며 promoter II의 크기와 효소활성은 4.0 Kb와 168.8 U/mg이었다. 형질 전환체에서의 YEp plasmid 안정성은 52.7%에서 67.4% 정도였다. YEp plasmid로부터 YIp plasmid를 재조합하였으며 이 YIp plasmid는 대장균에서나 효모에서도 발현되었다. 효모로부터 분리한 promoter I과 II는 재조합된 YEp와 YIp plasmid의 promoter로서 이용 가능하였다.

• 한국미생물·생명공학회지
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• 제30권3호
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• pp.206-209
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• 2002

Bacillus stearothermophilus의 CCTase 유전자(cgtS) 대장균과 효모의 shuttle vector로서 항구적 promoter인 adh l promoter를 함유한 pVT103-U(6.9Kb)에 도입하여 재조합 plasmid pVT-CCTS (9.0Kb)을 구축하고 효모 숙주 S. cerevisiae 2805에서 발현시켰다. 재조합 균주의 항구적 발현계인 2805/pv7-CGTS의 최적 발현조건은 YP배지에 dextrose 2%, pH 5.5, 30"C에서 최적 발효조건이었으며, CCTase의 최대 발현량은 48시간 배양시 0.624unit/mL을 나타내었다. B. stearothermophilus의 signal peptide가 재조합 효모에서도 높은 분비효율을 나타내어서 발현된 효소의 87%가 세포 외로 분비 생산되었다.산되었다.

• Journal of Microbiology and Biotechnology
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• 제21권8호
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• pp.830-837
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• 2011

A genomic DNA fragment encoding a putative maltooligosyltrehalose trehalohydrolase (NfMTH) for trehalose biosynthesis was cloned by the degenerate primer- PCR from cyanobacterium Nostoc flagelliforme. The ORF of NfMTH is 1,848 bp in length and encodes 615 amino acid residues, constituting a 70 kDa protein. The deduced amino acid sequence of NfMTH contains 4 regions highly conserved for MTHs. By expression of NfMTH in E. coli, the function of this protein was demonstrated, where the recombinant protein catalyzed the hydrolysis of maltooligosyl trehalose to trehalose. The expressions of MTH and maltooligosyltrehalose synthase in the filaments of N. flagelliforme were upregulated significantly under dehydration stress, NaCl stress, and high temperature-drought stress. The accumulations of both trehalose and sucrose in the filaments of N. flagelliforme were also improved significantly under the above stresses. Furthermore, trehalose accumulated in smaller quantities than sucrose did when under NaCl stress, but accumulated in higher quantities than sucrose did when under temperature-drought stress, indicating that both trehalose and sucrose were involved in N. flagelliforme adapted to stresses and different strategies conducted in response to various stress conditions.

• Animal cells and systems
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• 제11권2호
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• pp.175-182
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• 2007

The lysozyme II gene of cabbage butterfly Artogeia rapae was cloned from fat body of the larvae injected with E. coli and its nucleotide sequence was determined by the RACE-PCR. It has an open reading frame of 414 bp nucleotides corresponding to 138 amino acids including a signal sequence of 18 amino acids. The estimated molecular weight and the isoelectric point of the lysozyme II without the signal peptide were 13,649.38 Da and 9.11, respectively. The A. rapae lysozyme II (ARL II) showed the highest identity (81%) in the amino acid sequence to Manduca sexta lysozyme among other lepidopteran species. The two catalytic residues ($Glu^{32}$ and $Asp^{50}$) and the eight Cys residue motifs, which are highly conserved among other c-type lysozymes in invertebrates and vertebrates, are also completely conserved. A phylogenetic analysis based on amino acid sequences indicated that the ARL II was more closely related to M. sexta, Hyphantria cunea, Heliothis virescens, and Trichoplusia ni lysozymes. The ARL II gene was expressed in Spodoptera frugiperda 21 insect cells and the recombinant ARL II (rARL II) was purified from cell-conditioned media by cation exchange column chromatography and reverse phase FPLC. The purified rARL II was able to form a clear zone in lysoplate assay against Micrococcus luteus. The lytic activity was estimated to be 511.41 U/mg, 1.53 times higher than that of the chicken lysozyme. The optimum temperature for the lytic activity of the rARL II was $50^{\circ}C$, the temperature dependency of the absolute lytic activity of rARL II was higher than that of the chicken lysozyme at low temperatures under $65^{\circ}C$.

• KSBB Journal
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• 제20권4호
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• pp.291-298
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• 2005

본 연구는 재조합 대장균과 S.cerevisiae의 발효공정에서 형광스펙트럼 데이터를 수집하였으며, SOM을 이용하여 형광스펙트럼 데이터를 특정 그룹으로 분류하고 발효공정을 분석하고자 하였다. 배출가스 내 이산화탄소농도와 세포농도 같은 공정변수들은 SOM 알고리즘으로부터 얻은 분산 및 정규화된 가중치들과 좋은 연관성을 나타내었다. 전체 스펙트럼 데이터의 분류는 생물공정 모델링을 위한 매우 중요한 단계인데 그 이유는 몇몇 여기파장과 방출파장의 유의한 조합들이 전체영역의 스펙트럼 데이터로부터 추출되기 때문이다. 예를 들면, 본 연구에서 SOM을 이용하여 추출한 98개의 스펙트럼 데이터의 예제들은 부분최소자승법이나 감독신경망 (supervised neural network)을 이용한 공정의 모델링에 사용될 수 있다.

• 생약학회지
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• 제40권3호
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• pp.238-243
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• 2009

We examined ethanol extracts prepared from 23 Korean local plants obtained in Pyeongchang, Gangwon-do for their inhibitory effects on recombinant human AKR 1 B10 (rhAKR1B10) in vitro. To do this, rhAKR1B10 was first expressed in E. coli as a biological active form and purified by using Ni-affinity chromatography followed by gel permeation chromatography. Then, rhAKR1B10 was used for screening out 23 Korean local plant extracts having an inhibitory activity against itself. Among them, six extracts showed more than 50% inhibition of rhAKR1B10 activity at the concentration of $10{\mu}g$/ml. Especially, the extracts of Ligularia fischeri var. spiciformis Nakai and Rhus trichocarpa Miq. were the most potent because their $IC_{50}$ values were 2.94 and $2.00{\mu}g$/ml, respectively.

• 대한바이러스학회지
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• 제27권2호
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• pp.115-128
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• 1997

Three cDNA fragments located within NS5 region of HCV were synthesized by RT using viral RNA extracted from blood sample of Korean patient as a template. The cDNAs were amplified by PCR, cloned into the T-vector, and the nucleotide sequences were determined. Comparative analysis of the nucleotide and amino acid sequence of NS5 cDNAs showed that it is closely related with HCV type 1b. The cloned NS5 cDNA showed 91-94% homology at the nucleotide sequence level and 96-98% homology at the amino acid sequence level with several strains of the HCV type 1b. The NS5 cDNAs were subcloned into E. coli expression vectors to construct pRSETA5-1, pTHAN5-1, pRSETC5-2, pRSETBB1, pRESTCB1 and pRSETB-H3. Expression of the NS5 proteins was achieved by inducing the promoter with isopropyl-thio-${\beta}$-D-galactoside (IPTG) and confirmed by SDS-polyacrylamide gel electrophoresis. The NS5 proteins were immunoreactive against sera from Korean hepatitis C patients in Western blot analysis. Among the recombinant NS5 proteins, pRSETAS-1 plasmid derived protein, coded from aa2022 to aa2521 of HCV polyprotein, showed the strongest immunoreactivity against sera from Korean hepatitis C patients in immunoblot analysis. These results suggest that NS5 proteins would be useful as an antigen for detection of antibody against HCV in the blood samples.

• Asian-Australasian Journal of Animal Sciences
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• 제21권9호
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• pp.1286-1295
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• 2008

Leptin is produced by adipocytes and its role in the regulation of lipid metabolism, feed intake, productive and reproductive performance of domestic animal species has been greatly stressed and extensively investigated in recent years. This study was conducted to develop a radioimmunoassay (RIA) for the estimation of plasma bovine leptin and to determine plasma leptin concentration in fattening Japanese Black cattle (Wagyu) and its crossbreds at commercial farms. Relationships of plasma leptin with plasma vitamin A and age of crossbred cattle were also determined. Recombinant bovine leptin (rbleptin) was produced by the E. coli overexpressed leptin as a GST (glutathione S-transferase)-fusion protein. Then antiserum against bovine leptin was obtained by its immunization in rabbits. Using this antiserum, a bovine specific RIA was developed and plasma leptin level was determined in 120 crossbred fattening cattle (WagyuHolstein, 50:50) at commercial farms. The plasma leptin level increased with the age of cattle and its level was greater in the crossbred heifers than in the steers. Plasma vitamin A level was negatively correlated with plasma leptin level in crossbred heifers and steers. This relationship was stronger in heifers than in steers. Plasma leptin was gradually increased with advancing age in fattening Wagyu cattle. In conclusion, development of a bovine specific RIA to estimate plasma leptin will contribute to better understanding of the role of leptin in cattle.

• BMB Reports
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• 제31권2호
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• pp.111-116
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• 1998

HLA-DR4 is the dominant allele of MHC class II genes in Koreans. In particular, the $DRB1^*0405$ subtype has been reported to be almost exclusively expressed in Far East Asians, and has also been observed to be strongly associated with rheumatoid arthritis in Koreans and the Japanese. Identification of this specific allele has been mainly performed by PCR-based methods, which is often time consuming, costly, and involves tedious procedures such as the isolation of genomic DNA, PCR, and gel electrophoresis. To develop a more convenient tool for screening vast amounts of samples as well as to generate reagents which might also be used in other applications, in this study, antibodies were produced against this specific HLA subtype. By PCR, an allelespecific region covering the ${\beta}1$ domain of $DRB1^*0405$ was amplified and recombinantly expressed in E.coli. Immunization of Lewis rats with the purified protein yielded an allele specific antiserum. Western blot analysis showed the selective detection of the HLA-DR ${\beta}-chain$. Using this antiserum, established cell lines and peripheral blood lymphocytes were analyzed on their HLA haplotype by fluorescence activated flow cytometry. These novel antibodies will provide a powerful tool in the detection and investigation of DR4 alleles.