Stress Adaptation of Escherichia coli as Monitored via Metabolites by Using Two-Dimensional NMR Spectroscopy

채영기,김설현 한국자기공명학회 2017 Journal of the Korean Magnetic Resonance Society Vol.21 No.3

Escherichia coli responds to ever-changing external and internal stresses by rapidly adjusting its physiology for better survival. This adjustment occurs at all levels including metabolites as well as mRNAs and proteins. Although there has been many reports describing E. coli’s adaptation to various stresses regarding transcriptomics or proteomics, only a few investigations have been reported regarding this adaptation viewed from metabolites’ perspective. We applied four different types of stresses at four different doses as imposed by NaCl, sorbitol, ethanol, and pH to investigate the similarities or differences among the stresses, and which stress causes the largest perturbation of the metabolite composition. We profiled the metabolites under such external stresses by using two-dimensional NMR spectroscopy and identified 39 metabolites including amino acids, sugars, organic acids, and nucleic acids. According to our statistical analysis, the osmotic stress caused by sorbitol differentiated itself from others, while NaCl showed the largest dose dependent metabolic perturbations. We hope this work will form a foundation on which an approach to a successful protein production is systematically provided by a favorable metabolic environment by imposing proper external stresses.

Relationship between Protein Expression Pattern and Host Metabolome Perturbation as Monitored by Two-Dimensional NMR Spectroscopy

채영기,김설현,엄윤진 대한화학회 2019 Bulletin of the Korean Chemical Society Vol.40 No.7

In producing large amounts of heterologous proteins, researchers most often use Escherichia coli as a host thanks to its extensively studied genetics, simple growth procedure, and low cost. However, the desired protein is often produced only in a form of inclusion bodies. Researchers have tried to devise a way to circumvent such a problem, and the ones using fusion partners seem to be the most successful. Based on our previous observation that the host metabolome was related to the outcomes of protein expression patterns, we proceeded to perturb the metabolome by applying a salt stress to see if we could shake up metabolite compositions to make them better suited for soluble expression of the target protein. We examined a subset of the metabolites which had been partially labeled with input 13C-glucose. We tested 11 genes under 4 different NaCl concentrations, and identified 18 metabolites using the heteronuclear single quantum coherence NMR experiment. Most of the proteins kept their expression profiles unchanged, but two proteins were converted from inclusion bodies to a soluble form with increased NaCl concentration. Through the statistical analysis, we could identify a region where the soluble protein production was favored in the metabolite space. We hope that this work would provide an alternative strategy to produce the recombinant proteins in their soluble or native forms, not only in E. coli but also in other hosts.

Searching for Growth Conditions for Optimized Expression of Recombinant Proteins in Escherichia coli by Using Two-Dimensional NMR Spectroscopy

채영기,김설현 대한화학회 2015 Bulletin of the Korean Chemical Society Vol.36 No.1

Escherichia coli has been widely used as a host to produce recombinant proteins. However, because of its prokaryotic nature, it often fails to produce eukaryotic proteins in their native or correctly folded forms. To devise a systematic way to find such a condition that produces a large amount of correctly folded proteins, we chose to perturb the composition of intracellular metabolites by imposing different stresses such as the addition of sorbitol, NaCl, pH, and ethanol, and to monitor which metabolites showed the largest differences in the case of successful protein production. We profiled the metabolites under stresses by using two-dimensional NMR spectroscopy, and identified 39 metabolites after the protein production phase. We found that mild NaCl stress increased the yield of the target protein. Mildly high pH stress also helped in producing more of the target protein, and there is likely a connection between the isoelectric point of the target protein and the pH of medium for a higher yield. Exogenous sorbitol appeared to have been used to produce an osmoprotectant (trehalose) or direct energy source (glucose); thus it is advised that its use should not include isotopic labeling. Mild ethanol stress did not cause any significant change either in the metabolite profile or in the target protein production. We hope this work might shed light on how to improve production of the target protein by adjusting the external environment.

Discrimination of Rice Products by Geographical Origins and Cultivars by Two-Dimensional NMR Spectroscopy

채영기,김설현 대한화학회 2016 Bulletin of the Korean Chemical Society Vol.37 No.10

Rice is one of the most important crops that feed almost half of the world's population. With the increasing concern of consumers on the integrity of the product, efforts have been made to develop analytic techniques to discriminate rice products according to their origins or cultivars, but those efforts were mostly based on elemental analysis. We postulated that such discrimination would be possible with the global metabolite profiles. Nineteen metabolites of three different rice cultivars from four different geographical origins were identified from the extracts and compared with one another by the two-dimensional nuclear magnetic resonance (NMR) spectroscopy. NMR data were analyzed with the help of the metabolome database and the statistics software. The different rice samples were successfully separated in the principal component space, showing that the global metabolite profiles can be used to discriminate geographic origins. Our results show that the metabolite analysis via1H–13C heteronuclear single quantum coherence spectra combined with the statistical method can be applied to discriminate the geographic origins or cultivars of rice samples, thus can provide a means to inspect and pick up fraudulent labeling or adulteration.

메이에르홀드의 『발라간칙』: 새로운 연극을 향한 메이에르홀드의 시도

전명선,김설현 중앙대학교 외국학연구소 2013 외국학연구 Vol.- No.25

An interest to the theater, which began from the 20th century’s Russian Symbolism, served as a momentum to explore the identity of the Russian drama theater. But the ideal of Symbolism caused, as an anti-these of it, Blok’s “pathos” in his drama 『Balaganchik』. By directing Blok’s 『Balaganchik』Meyerhold finds not only the theater technique as a type of theater but also forms of anti-reproduce plays against Stanislabski. The elements of farce in Block’s 『Balaganchik』 provided Meyerhold with a source of theatrical techniques. It includes the role of masks, which formalizes the figures, making the play unfixed in speech of actors and the structure of play-within-a-play, also the grotesque originated from a romantic irony. All of these theatrical techniques were the self-reflective type of theater, presupposing the theatrical conditions. The conditions of theater are not just a cause of failure of the Realism in theater but also the ‘keyword’ of Meyerhold’s theater. And it is a part of effort to establish ontological status of Art in the background of the 20th century’s Modernism. Meyerhold’s 『Balaganchik』 reveals the overall condition of the theater, and stimulates the imagination of the audience by metaphorically depicting an old structure of society. And the concept of the grotesque, as the result of these experiments, provides a blueprint of Meyerhold’s theater which was conducted forth by him.

Memristor 특성에 따른 MLP(Multi-layer perceptron) 학습률의 변화

김예빈(Yebin Kim),김경진(Gyeongjin Kim),김민지(Minji Kim),김설현(Seolhyeon Kim),염지윤(Jiyoon Yeom),이종환(Jonghwan Lee) 대한전기학회 2023 대한전기학회 학술대회 논문집 Vol.2023 No.11