• Journal of Microbiology and Biotechnology
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• 제27권7호
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• pp.1345-1358
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• 2017

The impact of overproduction of a heterologous protein on the metabolic system of host Lactococcus lactis was investigated. The protein expression profiles of L. lactis IL1403 containing two near-identical plasmids that expressed high- and low-level of the green fluorescent protein (GFP) were examined via shotgun proteomics. Analysis of the two strains via high-throughput LC-MS/MS proteomics identified the expression of 294 proteins. The relative amount of each protein in the proteome of both strains was determined by label-free quantification using the spectral counting method. Although expression level of most proteins were similar, several significant alterations in metabolic network were identified in the high GFP-producing strain. These changes include alterations in the pyruvate fermentation pathway, oxidative pentose phosphate pathway, and de novo synthesis pathway for pyrimidine RNA. Expression of enzymes for the synthesis of dTDP-rhamnose and N-acetylglucosamine from glucose was suppressed in the high GFP strain. In addition, enzymes involved in the amino acid synthesis or interconversion pathway were downregulated. The most noticeable changes in the high GFP-producing strain were a 3.4-fold increase in the expression of stress response and chaperone proteins and increase of caseinolytic peptidase family proteins. Characterization of these host expression changes witnessed during overexpression of GFP was might suggested the metabolic requirements and networks that may limit protein expression, and will aid in the future development of lactococcal hosts to produce more heterologous protein.

• 정보처리학회논문지D
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• 제15D권5호
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• pp.681-690
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• 2008

단백질체학에서 특정 조건 하에서 단백질의 기능 이상 및 구조 변형 유무를 규명하고 질병 과정을 추적하는 것은 중요한 연구이다. 일반적으로 단백질의 발현량 변화 분석에는 통계적 방법이 많이 사용되고 있으며 단백질 상용 이미지 분석 소프트웨어에서 제공하는 그래픽을 이용한 방법들도 있으나, 이 방법들은 많은 조직 내에 존재하는 수많은 단백질을 수동으로 비교해야 하는 어려움이 있다. 본 논문에서는 데이터베이스와 데이터마이닝 기법을 이용하여 OLAP 데이터 큐브와 Discovery-driven 탐색의 응용 방법을 제안한다. 데이터 큐브의 특성을 이용함에 의해서, 질병에 의해 발현량이 변하는 단백질 뿐 아니라 임상적 특성과 단백질의 영향 관계를 분석하는 것이 가능하다. 데이터 큐브에서 단백질의 발현량 변화 분석에 적합한 데이터 큐브의 척도와Discovery-driven 탐색을 위한 예외 지표를 제안하고, 특히 In-exception을 계산하는데 있어서의 계산량 감소 방안을 제시한다. 실험을 통해 폐암 2-DE 데이터에서 데이터 큐브와 Discovery-driven 방법이 유용함을 보인다.

• 한국축산식품학회지
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• 제30권3호
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• pp.385-391
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• 2010

This study was conducted to compare proteins expressed in M. longissimus from Hanwoo and Holstein steers immediately after slaughter. Two-dimensional electrophoresis (2DE)/LC-MS/MS analysis revealed that the total number of detectable protein spots from longissimus muscle tissues was slightly higher in Hanwoo ($575{\pm}65$) than Holstein ($534{\pm}13$) steers, but that these numbers were not statistically significant due to large variation between replicates. A total of twelve protein spots did not match between sample groups, eight of which were expressed in the Hanwoo sample and four that were expressed in the Holstein sample. The protein spots detected in the Hanwoo sample included smooth muscle and non-muscle myosin alkali light chain 6B isomers, ${\alpha}B$ crystallin isomers, hemoglobin ${\beta}$-A chains, slow myosin heavy chains, and slow skeletal muscle troponin T chains. Collectively, these proteins are a class of slow-twitch muscle fiber and mirror that Hanwoo muscle tissue sampled for the current study contained more slow-twitch muscle fibers than Holstein one. Conversely, proteins detected from the Holstein sample included ankyrin repeat domain 2 and creatin kinase isomers. Given that creatin kinase isomers are related to the fast-twitch muscle, these results likely indicate that Holstein muscle tissue sampled for the current study contained more fast-twitch muscle fibers than Hanwoo beef.

• Journal of Forest and Environmental Science
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• 제26권2호
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• pp.83-94
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• 2010

Chinese fir (Latin name: Cunninghaimia lanceolata) is one of the major commercial coniferous trees. Most of Chinese fir forests are managed in successive rotation sites, which lead productivity to decline. Autotoxicity is the important reason for soil degradation of Chinese fir plantation, especially, phenolic acids are considered as the major allelopathic toxins which induce autotoxicity in Chinese fir rotation stands. We performed here proteomic approach to investigate the response of proteins in Chinese fir leaves to salicylic acid. The tube plantlets of Chinese fir clone were treated with 120 mg/L salicylic acid for 1, 3 and 5th day. 2-DE, coupled with MALDI-TOF-TOF/MS, was used to separate and identify the responsive proteins. We found 12, 7, and 12 candidate protein spots that were up- or down-regulated by at least 2.5 fold after 1, 3, and 5th day of the stress, respectively. Of these protein spots, 16 spots were identified successfully. According to the putative physiological functions, these proteins were categorized into five classes (1) the proteins involved in protein stability and folding, including 26S proteome, Grp78, Hsp70, Hsp90 and PPIase; (2) the protein involved in photosynthesis and respiration, including OEC 33 kDa subunit, GAPDH; (3) the protein related to cell endurance to acid, F-ATPase; (4) the protein related to cytoskeleton, tubulin; (5) the protein related to protein translation: prolyl-tRNA synthetase. These results give new insights into autotoxic substance stress response in Chinese fir leaves and provide preliminary footprints for further studies on the molecular signal mechanisms induced by the stress.

• Journal of Veterinary Science
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• 제23권1호
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• pp.3.1-3.12
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• 2022

Background: Domestic yaks are the most important livestock species on the Qinghai-Tibetan Plateau. Adult female yaks normally breed in the warm season (July to September) and enter anestrous in the cold season (November to April). Nevertheless, it is unclear how ovarian activity is regulated at the molecular level. Objectives: The peculiarities of yak reproduction were assessed to explore the molecular mechanism of postpartum anestrus ovaries in yaks after pregnancy and parturition. Methods: Sixty female yaks with calves were observed under natural grazing in Haiyan County, Qinghai Province. Three yak ovaries in pregnancy and postpartum anestrus were collected. RNA sequencing and quantitative proteomics were employed to analyze the pregnancy and postpartum ovaries after hypothermia to identify the genes and proteins related to the postpartum ovarian cycle. Results: The results revealed 841 differentially expressed genes during the postpartum hypoestrus cycle; 347 were up-regulated and 494 genes were down-regulated. Fifty-seven differential proteins were screened: 38 were up-regulated and 19 were down-regulated. The differential genes and proteins were related to the yak reproduction process, rhythm process, progesterone-mediated oocyte maturation, PI3K/AKT signaling pathway, and MAPK signaling pathway categories. Conclusions: Transcriptome and proteomic sequencing approaches were used to investigate postpartum anestrus and pregnancy ovaries in yaks. The results confirmed that BHLHE40, SF1IX1, FBPX1, HSPCA, LHCGR, BMP15, and ET-1R could affect postpartum hypoestrus and control the state of estrus.

• 한국작물학회:학술대회논문집
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• 한국작물학회 2022년도 추계학술대회
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• pp.191-191
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• 2022

Heterostyly continues to fascinate evolutionary biologists interested in heredity, evolution, breeding, and adaptive function. Polymorphism demonstrates how simply inherited developmental changes in the location of plant sexual associations can have important consequences for population pollination and mating biology. In contrast to homozygous self incompatibility, only a small number of mating phenotypes can be maintained in the population because insect pollinators have limitations in achieving multiple segregation sites for pollen deposition. Field studies of pollen tube growth have shown that reciprocal style-stamen polymorphisms function to increase the capacity of insect-mediated cross-pollination. The genetic pattern of style morphs is well established in various taxa, but despite recent advances, the identity, number, and structure of the genes controlling the heteromorphic syndrome have been poorly elucidated. The phenomenon of heterostyly in buckwheat has been controlled by gene complex concentrate to S-locus. Homomorphic autogamous buckwheat strains were established by the interspecific hybridization. Backcrossing of this line to the common buckwheat (pin) and selecting homostylar progenies made it possible to introduce the self-compatible gene into common buckwheat. In the result, we obtained the BC₉F₂ generation, and defined the strong linkage between flower type and self-incompatibility by microscopic observation of pollen tube growth. This finding suggests that self-incompatibility character is not controlled by one gene. Moreover, we defined the strong linkage between flower type and self-incompatibility. It strongly supports the S supergene theory. Therefore, we have plan to elucidate the heterostyly self-incompatibility by using molecular genetics, proteome analysis and apply to exploitation of buckwheat improvement. In near future, the expression of heterozygous syndromes in genus Fagopyrum with single isolated heterozygous species may provide clues to early stages of polymorphic assembly and shed light on evolutionary models of heterozygous strains.

• Genomics & Informatics
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• 제21권3호
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• pp.42.1-42.23
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• 2023

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, one of the most deadly infections in humans. The emergence of multidrug-resistant and extensively drug-resistant Mtb strains presents a global challenge. Mtb has shown resistance to many frontline antibiotics, including rifampicin, kanamycin, isoniazid, and capreomycin. The only licensed vaccine, Bacille Calmette-Guerin, does not efficiently protect against adult pulmonary tuberculosis. Therefore, it is urgently necessary to develop new vaccines to prevent infections caused by these strains. We used a subtractive proteomics approach on 23 virulent Mtb strains and identified a conserved membrane protein (MmpL4, NP_214964.1) as both a potential drug target and vaccine candidate. MmpL4 is a non-homologous essential protein in the host and is involved in the pathogen-specific pathway. Furthermore, MmpL4 shows no homology with anti-targets and has limited homology to human gut microflora, potentially reducing the likelihood of adverse effects and cross-reactivity if therapeutics specific to this protein are developed. Subsequently, we constructed a highly soluble, safe, antigenic, and stable multi-subunit vaccine from the MmpL4 protein using immunoinformatics. Molecular dynamics simulations revealed the stability of the vaccine-bound Tolllike receptor-4 complex on a nanosecond scale, and immune simulations indicated strong primary and secondary immune responses in the host. Therefore, our study identifies a new target that could expedite the design of effective therapeutics, and the designed vaccine should be validated. Future directions include an extensive molecular interaction analysis, in silico cloning, wet-lab experiments, and evaluation and comparison of the designed candidate as both a DNA vaccine and protein vaccine.

• BMB Reports
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• 제45권4호
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• pp.253-258
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• 2012

The dermal papilla cells (DPCs) of hair follicles are known to secrete paracrine factors for follicular cells. Shotgun proteomic analysis was performed to compare the expression profiles of the secretomes of human DPCs and dermal fibroblasts (DFs). In this study, the proteins secreted by DPCs and matched DFs were analyzed by 1DE/LTQ FTICR MS/MS, semi-quantitatively determined using emPAI mole percent values and then characterized using protein interaction network analysis. Among the 1,271 and 1,188 proteins identified in DFs and DPCs, respectively, 1,529 were further analyzed using the Ingenuity Pathway Analysis tool. We identified 28 DPC-specific extracellular matrix proteins including transporters (ECM1, A2M), enzymes (LOX, PON2), and peptidases (C3, C1R). The biochemically-validated DPC-specific proteins included thrombospondin 1 (THBS1), an insulin-like growth factor binding protein3 (IGFBP3), and, of particular interest, an integrin beta1 subunit (ITGB1) as a key network core protein. Using the shotgun proteomic technique and network analysis, we selected ITGB1, IGFBP3, and THBS1 as being possible hair-growth modulating protein biomarkers.

• 대한수의학회지
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• 제42권2호
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• pp.231-239
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• 2002

Proteomics is an emerging powerful tool in studying protein expression and function. At present study, proteomics was employed to evaluate the antigenicity among Escherichia coli O157:H7 strains using 2-dimensional gel electrophoresis (2-DE) and immunoblotting, SDS-PAGE and immunblotting analysis revealed no big differences among E coli O157:H7 strains. 2-DE analysis, however, revealed common antigens as well as specific antigens. The immunoblotting analysis revealed 20 common antigenic spots among E coli O157:H7 strains. In addition, there were 3 and 13 spots as common antigens between ATCC 43894 and KSC 109, and between ATCC 43894 and ACH 5, respectively. Antigenic spots specific for individual strain were also identified as 15, 8 and 22 for ATCC 43894, ACH 5 and KSC 109, respectively. The common antigens would be useful by employing either vaccine development or diagnosis marker, or both, whereas the specific antigens of individual strains would be applicable for epidemiological study. This study suggest that proteome analysis, representative as 2-DE, is valuable tool in exploring the E. coli antigenicity.

• Journal of Animal Science and Technology
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• 제45권5호
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• pp.731-738
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• 2003

근육의 생화학적 특성을 단백질 수준에서 분석하기 위하여 3원교잡종 돼지 3개체를 선발하고, white muscle은 longissimus dorsi muscle을 red muscle은 soleus muslce을 분리하여 분석에 이용하였다. 각 근육조직은 수용성, 불수용성 단백질 및 총단백질로 분리하여 추출하였고, 이차원전기영동 분석을 위하여 17cm 길이의 immobilized pH gradient strip (Bio-Rad, 3-10NL)과 12% acrylamide gel을 이용하여 전개하였다. 각각의 gel은 coomassie stain과 silver stain을 통하여 가시화 하였고, PDQuest software을 통하여 단백질 발현양상을 분석하였다. 하나의 gel에서 평균 600개 이상의 단백질 spot을 관찰하였으며, 반복실험을 통하여 white muscle과 red muscle간에 발현의 차이를 보이는 5개의 단백질 spot을 확인할 수 있었다. 5개의 spot 중 4개의 단백질은 측정된 분자량과 pI값이 troponin I, T 및 myoglobin의 수치값과 유사한 것으로 확인되었다. 그러나, 1개의 spot은 오차범위 내에서 유사한 단백질을 확인할 수 없었다. 5개의 단백질 spot중 1개(spot 1)는 white muscle에서, 4개의 spot(spot 2～5)은 red muscle에서 높게 발현됨을 확인하였으며, 특히 spot 4의 경우 white muscle 보다 red muscle에서 평균 14.6배 높게 발현됨을 확인하였다. 본 연구는 근육의 생화학적 특성을 이해하는데 중요한 기초 자료로 활용될 수 있으며, 앞으로 white muscle과 red muscle의 단백질 발현 분석을 통하여 단백질 수준에서의 생화학적 특성에 관한 연구가 충분히 진행되어야 할 것이다.