• Applied Microscopy
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• 제38권2호
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• pp.125-133
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• 2008

Mitochondria 내막의 cytochrome-c-oxidase는 세포의 에너지 생합성에 중요한 요소이며, 세포자멸사와 각종세포의 병리학적 현상과 밀접한 연관성이 있는 전자전달계효소로 알려져 있다. Porin 단백은 mitochondria 내막과 외막에 분포하는 효소단백으로 전자전달계효소 형성과 ATP 운반에 관여하는 것으로 알려져 있다. 따라서 면역현미경법을 사용하여 cytochrome-c-oxidase의 분포와 porin 단백과의 연관성을 확인하여 mitochondria의 cristae에 분포하는 cytochrome-c-oxidase의 형성과 변화를 알아보고자 하였다. Cardiac muscle tissue의 sarcoplasm에는 많은 수의 mitochondria가 분포하며, cytochrome-c-oxidase가 풍부한 mitochondria와 porin 단백이 풍부한 mitochondria로 구별되었다. Cytochrome-c-oxidase가 풍부한 mitochondria는 porin 단백이 빈약하고 porin 단백이 풍부한 mitochondria는 cytochrome-c-oxidase가 소량 포함되어 있는 것으로 관찰되었다. 심근조직의 부위에 따라 근형질에 분포하는 mitochondria에 cytochrome-c-oxidase가 풍부한 mitochondria와 porin 단백이 풍부한 mitochondria가 각각 상이하게 분포하였다. 이상의 결과로 미성숙 mitochondria는 많은 양의 porin 단백을 함유하여 근형질로부터 단백질 소단위를 mitochondria 막내로 운반하여 cytochrome-c-oxidase를 형성시키고 mitochondria가 성숙하면서 ATP를 운반할 최소한 양의 porin 단백만을 남기고 소멸되는 것으로 추측된다.

• 대한의생명과학회지
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• 제10권2호
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• pp.153-161
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• 2004

Antibiotic resistance is often associated with the production of inner membrane proteins (for example, AcrAB/TolC efflux pump) that are capable to extrude antibiotics, detergents, dyes and organic solvents. In order to evaluate the unknown MarB function of Escherichia coli, especially focused on the function of OmpF porin, several mutants were construted by T4GT7 transduction. MarA plays a major roles in mar (multiple antibiotic resistance) phenotype with AcrAB/TolC efflux pump in E. coli K-12. Futhermore, MarA decreases OmpF porin expression via micF antisense RNA. Expression of acrAB is increased in strains containing mutation in marR, and in those carrying multicopy plasmid expressing marA. MarB protein of E. coli K-12 showed its activity at OmpF porin & TolC protein as target molecule. Some paper reported MarB positively regulates OmpF function. MarA shows mar phenotype, and MarB along with MarA show decreased MIC through OmpF function. By this experiment, MarB could decrease MIC through the OmpF porin & TolC protein as target.

• Journal of Microbiology and Biotechnology
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• 제19권9호
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• pp.1034-1040
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• 2009

Porin proteins of Gram-negative bacteria are outer membrane proteins that act as receptors for bacteriophages and are involved in a variety of functions like solute transport, pathogenesis, and immunity. Salmonella enterica serovar Typhi (S. Typhi), a Gram-negative bacterium, is the causative agent of typhoid fever. Porins of S. Typhi have been shown to have a potential role in diagnostics and vaccination. In the present study, the major outer membrane proteins OmpF and OmpC from S. Typhi were cloned in pQE30UA vector and expressed in E. coli. The immunogenic nature of the recombinant porin proteins were evaluated by ELISA by raising hyperimmune sera in Swiss Albino mice with three different adjuvants (i.e., Freund's adjuvant and two human-compatible adjuvants like montanide and aluminium hydroxide gel) and proved to be immunogenic. The recombinant OmpF and OmpC generated in this work may be used for further studies for vaccination and diagnostics.

• Journal of Microbiology and Biotechnology
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• 제18권5호
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• pp.845-851
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• 2008

TolC is an outer membrane porin protein and an essential component of drug efflux and type-I secretion systems in Gram-negative bacteria. TolC comprises a periplasmic $\alpha$-helical barrel domain and a membrane-embedded $\beta$-barrel domain. TdeA, a functional and structural homolog of TolC, is required for toxin and drug export in the pathogenic oral bacterium Actinobacillus actinomycetemcomitans. Here, we report the expression of the periplasmic domain of TdeA as a soluble protein by substitution of the membrane-embedded domain with short linkers, which enabled us to purify the protein in the absence of detergent. We confirmed the structural integrity of the TdeA periplasmic domain by size-exclusion chromatography, circular dichroism spectroscopy, and electron microscopy, which together showed that the periplasmic domain of the TolC protein family fold correctly on its own. We further demonstrated that the periplasmic domain of TdeA interacts with peptidoglycans of the bacterial cell wall, which supports the idea that completely folded TolC family proteins traverse the peptidoglycan layer to interact with inner membrane transporters.

• 생명과학회지
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• 제18권1호
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• pp.84-90
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• 2008

충청남도 병천면 일대의 6곳의 토양시료를 채취하여 망간을 산화하는 균주들을 순수분리 하고, 이 중 망간 산화능이 가장 우수한 한 균주를 최종 선별하여 본 실험에 사용하였다. 최종 선별된 균주의 생리, 생화학적 특성을 조사하고, 16S rRNA 염기 서열분석 등을 통하여 동정한 결과 최종 선별된 균주는 Pseudomonas sp. MN5로 확인되었다. Pseudomonas sp. MN5은 fructose와 maltose를 제외한 다양한 당을 이용하지 못하였으며, 항생제인 kanamycin, chloramphenicol, streptomycin 그리고 tetracycline에는 높은 감수성을 보이고, 리튬, 망간, 바륨과 같은 중금속에 대해서는 mg/ml 단위의 높은 내성을 나타냈다. 그리고 Pseudomonas sp. MN5의 망간산화 최적 pH는 7.5이고, 망간산화 활성이 proteinase K와 가열처리를 한 시료에서 저해되었다. Pseudomonas sp. MN5가 생성하는 망간산화 단백질을 ammonium sulfate precipitation, HiTrap Q FF ion exchange chromatography 그리고 G3000sw $_{XL}$ gel filtration chromatography를 통해서 정제한 결과, 15 kDa, 46.7 kDa 그리고 63.5 kDa의 세종류의 manganese oxidizing protein가 확인되었고, 내부서 열과 N-말단 서열 분석 결과 Pseudomonas sp. MN5가 생성하는 망간산화 단백질은 외막의 porin 단백질인 것으로 추정되었다.

• Journal of Microbiology and Biotechnology
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• 제15권6호
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• pp.1214-1220
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• 2005

Decreased porin-mediated outer membrane penetration of hydrophilic antibiotics is a common mechanism of antibiotic resistance in Gram-negative bacteria. This study was undertaken to determine whether a null mutation in Pseudomonas putida would suppress porin synthesis, and therefore reduce the susceptibility of the organism to streptomycin, norfloxacin, and tetracycline. Inverse PCR amplification and double-stranded DNA sequencing were used to identify chromosomal genes carrying TnphoA'-1 inserts. Genome database available was used to identify putative homologue genes, one of which encodes protein with homology to domains of the MutS of P. putida, suggesting a crucial role in the multidrug resistance. Increased resistance to streptomycin, norfloxacin, and tetracycline might be due to accumulation of compensatory mutations. Either no growth or slow growth was observed in P. putida KH1027 when grown in minimal medium containing gluconate, glucose, or citrate; however, it is not clear whether the growth patterns contributed to the multidrug resistance.

• Applied Microscopy
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• 제30권3호
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• pp.241-248
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• 2000

전자현미경을 이용한 단백질 구조분석은 약 30여년간 발전되어 왔다. Bacteriorhodopsin과 porin 등의 near atomic resolution의 구조분석은 전자현미경의 발전과 진행을 보여주는 하나의 예로 설명될 수 있다. 전자현미경을 이용한 거대분자의 3차원 구조를 규명하기 위해 필요한 기본과정 즉, 시료준비, 자료수집과 자료처리 등에 대하여 토론하였다.

• 생명과학회지
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• 제18권11호
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• pp.1606-1611
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• 2008

Salmoenella를 포함한 그람 음성 세균의 외막 단백질들은 외부 환경 조건에 따라 그 발현에 변화가 생기는 경우가 많으며, 이렇게 발현된 외막 단백질들은 수송에 관여하는 통로, 외부 물질의 인지, 병원체의 부착인자 등 다양한 기능을 가진다. 본 연구에서는 이러한 외막 단백질 중 소수성 porin으로 알려진 OmpW 단백질에 주목하였다. ompW 유전자가 결손된 돌연변이주를 구축하여 CK10으로 명명하였으며, 야생주와 그 표현형적 특징을 비교한 결과 SDS에 대한 내성이 다소 증가함을 확인할 수 있었다. OmpW 특이적인 다클론성 항체를 조제하여 OmpW 단백질의 발현연구에 사용하였다. NaCl의 농도가 높아질수록 OmpW 단백질의 발현이 감소하였으며, OmpW의 발현이 최대인 조건은 NaCl이 배지에 첨가되지 않았을 경우이다. 따라서 OmpW는 Salmonella 균이 삼투환경에 대응하는데 관여할 것으로 예상되어 일차적으로 균의 생육을 조사한 결과, 다양한 삼투환경 하에서 Salmonella의 생육에는 OmpW 단백질의 결손이 큰 영향을 미치지는 않는 것을 확인하였다. 삼투환경 변화에 따른 OmpW 발현의 변화가 지니는 생물학적 의미는 앞으로 연구해야 할 숙제로 남는다.

• Journal of Microbiology and Biotechnology
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• 제24권6호
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• pp.854-861
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• 2014

The diagnosis of Brucella abortus is mainly based on serological methods using antibody against LPS, which has diagnostic problems. Therefore, to solve this problem, we evaluated two proteins of B. abortus, Cu/Zn superoxide dismutase (SodC) and outer membrane proteins 2b porin (Omp2b). The genes were cloned and expressed in a pMAL system, and the recombinant proteins, rOmp2b and rSodC, were purified as fusion forms with maltose-binding protein. The identity of the proteins was confirmed by SDS-PAGE and Western blot analysis with sera of mice infected with B. abortus. Production of cytokines and nitric oxide (NO) was investigated in RAW 264.7 cells and mouse splenocytes after stimulation with the proteins. Moreover, cellular and humoral immune responses were investigated in BALB/c mice after immunization with the proteins. TNF-${\alpha}$, IL-6, and NO were significantly inducible in RAW 264.7 cells. Splenocytes of naive mice produced IFN-${\gamma}$ and IL-4 significantly by stimulation. Moreover, number of IgG, IFN-${\gamma}$, and IL-4 producing cells were increased in immunized mice with the two proteins. Production of IgG and IgM with rOmp2b was higher than those with rSodC in immunized mice. These results suggest that the two recombinant proteins of B. abortus may be potential LPS-free proteins for diagnosis.

• International Journal of Oral Biology
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• 제42권2호
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• pp.63-70
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• 2017

Selecting an appropriate antigen with optimal immunogenicity and physicochemical properties is a pivotal factor to develop a protein based subunit vaccine. Despite rapid progress in modern molecular cloning and recombinant protein technology, there remains a huge challenge for purifying and using protein antigens rich in hydrophobic domains, such as membrane associated proteins. To overcome current limitations using hydrophobic proteins as vaccine antigens, we adopted in silico analyses which included bioinformatic prediction and sequence-based protein 3D structure modeling, to develop a novel periodontitis subunit vaccine against the outer membrane protein FomA of Fusobacterium nucleatum. To generate an optimal antigen candidate, we predicted hydrophilicity and B cell epitope parameter by querying to web-based databases, and designed a truncated FomA (tFomA) candidate with better solubility and preserved B cell epitopes. The truncated recombinant protein was engineered to expose epitopes on the surface through simulating amino acid sequence-based 3D folding in aqueous environment. The recombinant tFomA was further expressed and purified, and its immunological properties were evaluated. In the mice intranasal vaccination study, tFomA significantly induced antigen-specific IgG and sIgA responses in both systemic and oral-mucosal compartments, respectively. Our results testify that intelligent in silico designing of antigens provide amenable vaccine epitopes from hard-to-manufacture hydrophobic domain rich microbial antigens.