• 한국미생물·생명공학회지
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• 제39권1호
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• pp.20-28
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• 2011

두 개의 cellulase 유전자 xynB1, bglA2과 groEL1 유전자 염기서열에 기초하여 국내에서 분리한 34균주의 Sorangium cellulosum 균주들을 계통 분석한 결과, 점액세균 중 가장 많은 생리활성물질이 발견된 종인 S. cellulosum 내에는 최소한 5그룹의 소그룹이 존재함을 보였다. 이 분석은 또한 S. cellulosum 균주들의 다양성을 보여주어 분석한 34균주 중 30균주가 서로 다른 균주인 것으로 나타났다.

• Journal of Microbiology and Biotechnology
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• 제8권5호
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• pp.509-516
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• 1998

A plasmid vector, pXGPRMATG-lac-Tgx, was developed for overproduction of $\beta$-galactosidase in Escherichia coli using the genetic components of groEx, a heat-shock gene cloned from symbiotic X-bacteria in Amoeba proteus. The vector is composed of intragenic promoters P3 and P4 of groEx, the structural gene of lac operon, transcription tenninator signals of lac and groEx, and ColEl and amp'of pBluescript SKII. The optimized host, E. coli DH5$\alpha$, transfonned with the vector constitutively produced 117,310-171,961 Miller units of $\beta$-galactosidase per mg protein in crude extract. The amount of enzyme in crude extract was 53% of total water-soluble proteins. About 43% of the enzyme could be purified to a specific activity of 322,249 Miller units/mg protein after two-fold purification, using two cycles of precipitation with ammonium sulfate and one step of gel filtration. Thus, the expression system developed in this study presents a low-cost and simple method for purifying overproduced $\beta$-galactosidase in E. coli.

• Journal of Microbiology and Biotechnology
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• 제17권7호
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• pp.1177-1182
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• 2007

Bacillus cereus group bacteria share a significant degree of genetic similarity. Thus, to differentiate and identify the Bacillus cereus group efficiently, a multiplex PCR method using the gyrB and groEL genes as diagnostic markers is suggested for simultaneous detection. The assay yielded a 400 bp amplicon for the groEL gene from all the B. cereus group bacteria, and a 253 bp amplicon from B. anthracis, 475 bp amplicon from B. cereus, 299 bp amplicon from B. thuringiensis, and 604 bp amplicon from B. mycoides for the gyrB gene. No nonspecific amplicons were observed with the DNA from 29 other pathogenic bacteria. The specificity and sensitivity of the B. cereus group identification using this multiplex PCR assay were evaluated with different kinds of food samples. In conclusion, the proposed multiplex PCR is a reliable, simple, rapid, and efficient method for the simultaneous identification of B. cereus group bacteria from food samples in a single tube.

• Fisheries and Aquatic Sciences
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• 제16권4호
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• pp.273-277
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• 2013

Vibriosis caused by Vibrio anguillarum and edwardsiellosis caused by Edwardsiella tarda are septicemic diseases of many commercially important freshwater and marine fishes, and threaten the aquaculture industry in Korea. Early diagnosis and accurate identification of these two bacterial species could help to prevent these diseases and minimize the damage to cultured marine species. This study designed a duplex polymerase chain reaction (PCR) method for the simultaneous detection of two major fish pathogens: V. anguillarum and E. tarda. Each pair of oligonucleotide primers exclusively amplified the target groEL gene of the specific microorganism. Twenty-two Vibrio and ten non-Vibrio enteric species were used to check the specificity of the primers, which were found to be highly specific for the target species, even among closely related species. The detection limit was 400 pg for V. anguillarum and 4 ng for E. tarda when mixed purified DNA was used as the template. This assay showed high specificity and sensitivity in the simultaneous detection of V. anguillarum and E. tarda from artificially inoculated seawater and fish.

• 미생물학회지
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• 제37권3호
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• pp.221-227
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• 2001

열충격 단백질(heat shock protein: HSP)은 변성된 단백질의 응집을 방지하여 가혹한환경에서 병원균의 생존을 증가시킨다. 세균에 알코을 stress를 가하면 다량의 DnaK와 GronEL이 유도되지만 폐렴구균에서는 DnaK와 GroEL이 전혀 유도되지 않는 대신 알코올탈수소효소(alcohol dehydrogenase : ADH)가 유도되었다. 이런 특성은 폐렴구균 ADH가 HSP처럼 chaperone 기능을 수행라고 있을 가능성을 제시하고 있으므로 본 연구에서는 일차적으로 ADH 유전자를 확인하고 ADH 의 면역특성 및 세포내 분포를 측정하였다. 폐렴구균 ADH는 이질아메바 ADH2 및 대장균 ADH 와 높은 유사성을 나타냈으며 883 개의 아미노산으로 구성된 등전점 6.09의 단백질로 추정된다. 그러나 폐렴구균 ADH와 유사성이 높은 대장균, 유산균 및 황색포도상구균의 용해액을 폐렴구균 ADH 항체와 immunoblot을 실시하였을 때 전혀 반응하지 않았다. 또한 세포질, membrane, periplasm에 있는 단백질 분획 및 폐렴구균 배양 상등액을 ADH 항체와 immune blot을 실시하였을 때 ADH 는 열충격에 관계없이 세포 밖으로 분비되는 단백질임을 확인하였다. 이런 결과는 폐렴구균 ADH가 진단용항원 및 백신으로 개발될 수 있는 가능성을 제시하고 있다.

• Journal of Microbiology and Biotechnology
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• 제18권2호
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• pp.228-233
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• 2008

Effects of chaperones on mRNA stability and gene expression were studied in order to develop an efficient Escherichia coli expression system that can maximize gene expression. The stability of mRNA was modulated by introducing various secondary structures at the 5'-end of mRNA. Four vector systems providing different 5'-end structures were constructed, and genes encoding GFPuv and endoxylanase were cloned into the four vector systems. Primer extension assay revealed different mRNA half-lives depending on the 5'-end secondary structures of mRNA. In addition to the stem-loop structure at the 5'-end of mRNA, coexpression of dnaK-dnaJ-grpE or groEL-groES, representative heat-shock genes in E. coli, increased the mRNA stability and the level of gene expression further, even though the degree of stabilization was varied. Our work suggests that some of the heat-shock proteins can function as mRNA stabilizers as well s protein chaperones.

• Animal cells and systems
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• 제5권3호
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• pp.163-177
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• 2001

Most, if not all, aphids harbor intracellular bacterial symbionts, called Buchnera, in their bacteriocytes, huge cells differentiated for this purpose. The association between Buchnera and aphids is so intimate, mutualistic and obligate that neither of them can any longer reproduce independently. Buchnera are vertically transmitted through generations of the host insects. Evidence suggests that Buchnera were acquired by a common ancestor of aphids 160-280 million years ago, and have been diversified, since then, in parallel with their aphid hosts. Molecular phylogenetic analyses indicate that Buchnera belong to the g subdivision of the Proteobacteria. Although Buchnera are close relatives of Escherichia coli, they contain move than 100 genomic copies per cell, and their genome size is only one seventh that of E. coli. The complete genome sequence of Buchnera revealed that their gene repertoire is quite different from those of parasitic bacteria such as Mycoplasma, Rickettsia and Chlamydia, though their genome sizes have been reduced to a similar extent. Whereas these parasitic bacteria have lost most genes for the biosynthesis of amino acids, Buchnera retain many of them. In particular, Buchnera's gene repertoire is characteristic in the richness of the genes for the biosynthesis of essential amino acids that the eukaryotic hosts are not able to synthesize, reflecting a nutritional role played by these symbionts. Buchnera, when housed in the bacteriocyte, selectively synthesize a large amount of symbionin, which is a homolog of GroEL, the major stress protein of E. coli. Symbionin not only functions as molecular chaperone, like GroEL, but also has evolutionarily acquired the phosphotransferase activity through amino acid substitutions. Aphids usually profit from Buchnera's fuction as a nutritional supplier and, when faced with an emergency, consume the biomass of Buchnera cells as nutrient reserves.

• 한국자기공명학회논문지
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• 제17권1호
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• pp.30-39
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• 2013

Immortalization-upregulated protein-1 (IMUP-1) genes have been cloned and are known to be involved in SV40-mediated immortalization. IMUP-1 gene is highly expressed in various cancer cell lines and tumors, suggesting the possibility that they might be involved in tumorigenicity. Previously, there were several problems for overexpression of IMUP-1 in bacterial expression systems including low solubility and aggregation due to unstructured property. To investigate the structural properties, it is necessary to obtain lots of pure and soluble proteins. Accordingly, the co-expression systems of bacterial chaperone proteins, GroEL-GroES, were used to increase solubility of IMUP-1. From the analysis of NMR and CD experiment data, it is suggested that the protein adopt typical the random coil properties in solution.

• 한국미생물·생명공학회지
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• 제43권3호
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• pp.212-218
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• 2015

Fucosyltransferase는 퓨코실화된 올리고당을 생성하는데 필수적인 효소로서, GDP-β-L-fucose로 부터 fucose를 수용체로 전이시켜 알파 글리코사이드 결합을 형성하는 과정을 촉매한다. 하지만 Escherichia coli 에서 발현시켰을 때, 대부분의 경우 inclusion body를 형성하여 비활성으로 생성되었다. 따라서 본 연구에서는 Helicobacter pylori 26695에서 유래한 α1,2-fucosyltransferase (FucT2) 유전자의 수용성 발현을 위하여, E. coli에서 샤페론 단백질인 GroEL, GroES, DnaK, DnaJ, GrpE과 함께 동시발현시켰다. SDS-PAGE 분석결과, 5가지 샤페론 단백질과 함께 발현되었으며, 수용성 FucT2 단백질이 증가하였고 효소활성은 5배 증가하였다. 결론적으로, 샤페론 동시발현기술을 이용하여 대장균에서 FucT2 수용성 생산을 증가시킬 수 있었으며 본 수용성 효소는 퓨코실화된 올리고당을 효율적으로 생산하는데 이용될 수 있다.

• Journal of Microbiology and Biotechnology
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• 제28권5호
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• pp.739-747
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• 2018

The low solubility and high-cost recovery of Propionibacterium acnes polyunsaturated fatty acid isomerase (PAI) are key problems in the bioproduction of high value-added conjugated linoleic acid (CLA). To improve the solubility of recombinant PAI, six chaperone proteins were coexpressed with PAI. Introduction of GroELS proteins dramatically improved the PAI solubility from 29% to 97%, with increased activity by 57.8%. Combined expression of DnaKJ-GrpE and GroELS proteins increased the activity by 11.9%. In contrast, coexpression of DnaKJ-GrpE proteins significantly reduced the activity by 57.4%. Plasmids pTf16 harboring the tig gene and pG-Tf2 containing the tig and groEL-groES genes had no visible impact on PAI expression. The lytic protein E was then introduced into the recombinant Escherichia coli to develop a cell autolysis system. A 35% activity of total intracellular PAI was released from the cytoplasm by suspending the lysed cells in distilled water. The PAI recovery was further improved to 81% by optimizing the release conditions. The lipase from Rhizopus oryzae was also expressed in E. coli, with an extracellular activity of 110.9 U/ml. By using the free PAI and lipase as catalysts, a joint system was established for producing CLA from sunflower oil. Under the optimized conditions, the maximum titer of t-10,c-12-CLA reached 9.4 g/l. This work provides an effective and low-cost strategy to improve the solubility and recovery of the recombinant intracellular PAI for further large-scale production of CLA.