• 한국축산식품학회지
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• 제26권2호
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• pp.263-268
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• 2006

최근 들어 병원성 미생물의 저감화를 위하여 기존의 항생제 계열의 항균물질이 아닌 새로운 개념의 신소재 개발이 활발하게 진행 중에 있다. 특히, 이러한 신개념의 병원성 저감화 소재 중 인간의 장내에 존해하는 probiotics 균주의 특성을 이용하여 병원성 미생물을 예방하는 것은 보다 효과적인 방법 중의 하나가 될 수 있을 것으로 판단된다. 본 실험에서는 HT-29 cell을 대상으로 L. acidophilus 균체와 세포 파쇄물을 대상으로 STEC ATCC 43894의 장 상피세포 부착 억제능력을 측정하였다. 10 mg/mL의 세포 파쇄물이 존재하였을 때 $10^9cfu/mL$의 균체가 존재했을 때와 유사한 수준으로 STEC ATCC 43894의 부착 저해 효과가 관찰되었다. 하지만, L. acidophilus A4의 상등액에서는 그 저해 효과가 세포 파쇄물의 $5{\sim}10%$ 정도 수준으로 관찰되어 그 효과는 매우 적은 것으로 판단되었다. 또한, L. acidophilus A4의 세포 파쇄물이 STEC의 부착에 미치는 영향을 관찰하기 위하여 10mg/mL의 세포 파쇄물이 첨가된 배지에서 STEC의 단백질발현 양상을 확인하였다. 각 gel의 image에서 평균적으로 800개의 spot을 관찰할 수 있었으며 이중 2배 이상의 발현차이를 보이는 13개의 spot을 선발하였다. 7개의 spot은 세포파쇄물이 첨가되었을 때 발현이 증가하였으며 3개의 spot은 발현이 감소하였다. 흥미롭게도 3개의 단백질 spot은 세포파쇄물이 존재할 때만 발현되는 것을 확인하였다. 명확하지는 않지만 이러한 L. acidophilus A4의 세포 파쇄물에 존재하는 물질은 (1)STEC의 부착과 관련된 특정 단백질의 발현을 저해하거나 (2)STEC과 장상 피세포에서의 수용체 경합을 통해 부착을 억제하는 것으로 생각된다. 앞으로 이와 관련된 보다 세부적인 작용 메카니즘 연구 및 생화학적연구가 필요할 것으로 판단된다.

• 분석과학
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• 제19권6호
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• pp.529-534
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• 2006

SDS-PAGE를 이용한 일반적인 단백질 분리는 단백질을 분자량에 따라 분리하는 방법이며 가장 보편적이고, 간단한 방법 중의 하나이다. 본 연구는 SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis)를 두 번에 걸쳐 동일한 분리 원리로 이차원으로 전개하는 방법의 전기영동을 시도하여 기존의 일차원 전기영동 분석법과 비교하였으며, 그 결과 향상된 분리능이 본 연구의 이차원 전기영동법에서 확인되었다. Gel에서 분리된 단백질들은 MALDI TOF MS를 이용하여 동정하여 서로 다른 단백질임이 확인되었으며, 이러한 duplex SDS-PAGE 분리법은 상대적으로 적은 비용으로 단백질 분리능을 용이하게 향상시킬 수 있는 경제적인 분석법으로 이용될 수 있을 것이다.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 1986년도 추계학술대회
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• pp.516.2-516
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• 1986

DNA subfragments, sopA, sopB, and sopC supporting stable maintenance of an oriC plasmid, were derived from mini-F plasmid DNA (EcoRI restriction fragment, f5) after digestion with restriction endonucleases, and cloned in vector plasmid pBR322. The recombinant plasmid obtained were introduced into E. coli KY7231 and E. coli CSR603, and proteins specified by the mini-F fragments were analysed by SDS-polyacrylamide gel electrophoresis. Two proteins encoded by the F fragments were detected, having molecular weights of 41,000 and 37.000. The sopA protein (41K) encoded by a plasmid pXX288 was observed in the cytoplasm, whereas the sopB protein (37K) encoded by a plasmid pXX157 was in the membrane fraction. There was no novel protein band detected in the cell with a plasmid pXX300, which contained sopC fragment. Gene products of a plasmid pXX167, which is comprised of sopA, sopB, and sopC, were not detectable. Fluorography after one and two dimensional gel electrophoresis of the lysates showed that these two proteins were overproduced in the cells which were allowed to incorporate radioactive amino acid after plasmid amplification by chloramphenicol treatment. The isoelectric points of the sopA and sepB proteins were 6.6 and 7.0, respectively.

• Nutritional Sciences
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• 제9권4호
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• pp.317-322
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• 2006

Soybeans are well-known as allergenic foods. Koreans consume large amounts of soybean foods, such as kochujang, which have gone through the fermentation process. To lower the allergenicity of these foods, we prepared hypo allergenic kochujang with aloe extract (AK). A sensory evaluation was conducted along with a clinical evaluation that used a double-blind, placebo-controlled food challenge (DBPCFC) test These tests were designed to evaluate the acceptability of the fermented foods. In comparison to normal kochujang (NK), AK elicited a higher sensory test score, and the rate of positive reactions in atopic dermatitis patients during the DBPCFC test was reduced. Methods of protein extraction, protein quantitation with sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and protein identification using two-dimensional (2D) gel electrophoresis were performed for both NK and AK to compare the functional factors. We found a reduction in the levels of high molecular proteins even though the bands of the proteins had not entirely disappeared, indicating that the boiling and fermentation process changed the soybean protein patterns. The rate of the reduction of high molecular proteins was more effective in the AK. In conclusion, AK can be recognized as a food with hypoallergenic effect.

• Journal of Microbiology
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• 제44권4호
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• pp.369-374
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• 2006

To investigate the mechanism of salt tolerance of gram-positive moderately halophilic bacteria, two-dimensional gel electrophoresis (2-D PAGE) was employed to achieve high resolution maps of proteins of Halobacillus dabanensis $D-8^T$. Approximately 700 spots of proteins were identified from these 2-D PAGE maps. The majority of these proteins had molecular weights between 17.5 and 66 kDa, and most of them were distributed between the isoelectric points (pI) 4.0 and 5.9. Some protein spots were distributed in the more acidic region of the 2-D gel (pI <4.0). This pattern indicated that a number of proteins in the strain $D-8^T$ are acidic. To understand the adaptation mechanisms of moderately halophilic bacteria in response to sudden environmental changes, differential protein profiles of this strain were investigated by 2-D PAGE and $Imagemaster^{TM}$ 2D Platinum software after the cells were subjected to salt shock of 1 to 25% salinity for 5 and 50 min. Analysis showed 59 proteins with an altered level of expression as the result of the exposure to salt shock. Eighteen proteins had increased expression, S proteins were induced, and the expression of 33 proteins was down-regulated. Eight of the up-regulated proteins were identified using MALDI-TOF/MS and MASCOT, and were similar to proteins involved in signal transduction, proteins participating in energy metabolism pathways and proteins involved in stress.

• Journal of Microbiology and Biotechnology
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• 제22권4호
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• pp.470-478
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• 2012

Recent genome comparisons of E. coli B and K-12 strains have indicated that the makeup of the cell envelopes in these two strains is quite different. Therefore, we analyzed and compared the envelope proteomes of E. coli BL21(DE3) and MG1655. A total of 165 protein spots, including 62 nonredundant proteins, were unambiguously identified by two-dimensional gel electrophoresis and mass spectrometry. Of these, 43 proteins were conserved between the two strains, whereas 4 and 16 strain-specific proteins were identified only in E. coli BL21(DE3) and MG1655, respectively. Additionally, 24 proteins showed more than 2-fold differences in intensities between the B and K-12 strains. The reference envelope proteome maps showed that E. coli envelope mainly contained channel proteins and lipoproteins. Interesting proteomic observations between the two strains were as follows: (i) B produced more OmpF porin with a larger pore size than K-12, indicating an increase in the membrane permeability; (ii) B produced higher amounts of lipoproteins, which facilitates the assembly of outer membrane ${\beta}$-barrel proteins; and (iii) motility- (FliC) and chemotaxis-related proteins (CheA and CheW) were detected only in K-12, which showed that E. coli B is restricted with regard to migration under unfavorable conditions. These differences may influence the permeability and integrity of the cell envelope, showing that E. coli B may be more susceptible than K-12 to certain stress conditions. Thus, these findings suggest that E. coli K-12 and its derivatives will be more favorable strains in certain biotechnological applications, such as cell surface display or membrane engineering studies.

• Journal of Microbiology and Biotechnology
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• 제6권5호
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• pp.361-363
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• 1996

The terminal regions of the double-stranded RNA (dsRNA) genome segments of infectious pancreatic necrosis virus (IPNV) DRT strain were sequenced. The dsRNAs, which were $^{32}P$-labelled at their 3'-termini by incubation with [$^{32}P$]pCp and T4 RNA ligase, were separated by 5$%$ polyacrylamide gel electrophoresis, and the segments A and B of IPNV-DRT were sequenced by two-dimensional gel electrophoresis. The 5'-terminal sequences of the IPNV-DRT plus strand from two genome segments were found to have the same conserved nucleotide (5'-CGG(C/A)A-), but the 3'-terminal sequences -CCCCAGGCG-3' and -CGGACCCCG-3' were found in the plus strand from segments A and B, respectively. The inverted oligonucleotide sequences of 3'-terminal of between segments A and B were found and they differ from those of other IPNVs.

• Asian-Australasian Journal of Animal Sciences
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• 제28권6호
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• pp.788-795
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• 2015

Two dimensional-fluorescence difference gel electrophoresis (2D DIGE) is an emerging technique for comparative proteomics, which improves the reproducibility and reliability of differential protein expression analysis between samples. The purpose of this study was to investigate bovine pregnancy-specific proteins in the proteome between bovine pregnant and non-pregnant serum using DIGE technique. Serums of 2 pregnant Holstein dairy cattle at day 21 after artificial insemination and those of 2 non-pregnant were used in this study. The pre-electrophoretic labeling of pregnant and non-pregnant serum proteins were mixed with Cy3 and Cy5 fluorescent dyes, respectively, and an internal standard was labeled with Cy2. Labeled proteins with Cy2, Cy3, and Cy5 were separated together in a single gel, and then were detected by fluorescence image analyzer. The 2D DIGE method using fluorescence CyDye DIGE flour had higher sensitivity than conventional 2D gel electrophoresis, and showed reproducible results. Approximately 1,500 protein spots were detected by 2D DIGE. Several proteins showed a more than 1.5-fold up and down regulation between non-pregnant and pregnant serum proteins. The differentially expressed proteins were identified by MALDI-TOF mass spectrometer. A total 16 protein spots were detected to regulate differentially in the pregnant serum, among which 7 spots were up-regulated proteins such as conglutinin precursor, modified bovine fibrinogen and IgG1, and 6 spots were down-regulated proteins such as hemoglobin, complement component 3, bovine fibrinogen and IgG2a three spots were not identified. The identified proteins demonstrate that early pregnant bovine serum may have several pregnancy-specific proteins, and these could be a valuable information for the development of pregnancy-diagnostic markers in early pregnancy bovine serum.

• International Journal of Industrial Entomology and Biomaterials
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• 제9권1호
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• pp.117-121
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• 2004

The patterns of diapause-associated proteins of silkworm eggs were analyzed by two-dimensional (2-D) gel electrophoresis. Among the hundreds of spots on the 2-D gels, at least two proteins were considered to be associated with diapause. A protein, spot 4, with an approximate molecular weight of 38 kDa and pI 6.1 was observed in the HCI-treated, cold-treated, and diapause eggs, respectively. Spot 4 was undetectable in unfertilized eggs and non-diapause eggs at two days after oviposition, suggesting that this protein may be associated with the entrance to diapause. A protein, spot 11, with an approximate molecular weight of 21 kDa and pI of 61 was detected in the unfertilized, HCl-treated, and cold-treated eggs, respectively, after oviposition by normal moths. In diapausing eggs, a protein corresponding to spot 11 was observed in 3-, 5-, and 30-day-old eggs, while the protein was not detected one-day-old eggs. The protein corresponding to spot 11 was not detected in unfertilized and non-diapause eggs obtained from subesophabeal ganglion (SG)-extirpated moths either. Spot 11 was also considered to be a diapause specific protein, which occurred at only early embryonic stage under the control of diapause-downregulated gene.

• 한국작물학회지
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• 제49권spc1호
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• pp.307-317
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• 2004