• Journal of Applied Biological Chemistry
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• 제66권
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• pp.128-137
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• 2023

Transcriptome analysis revealed that the sinR gene encoding a transition-state regulator of Bacillus pumilus, genetically close to B. subtilis, was expressed at high levels during growth. The sinR gene is the second gene of the sinIR operon consisting of three promoters and two structural genes in B. subtilis. This study used the sinIR promoter of B. subtilis DB104 to construct a recombinant protein expression system. First, the expression ability depending on the number of sinIR promoter was investigated using enhanced green fluorescent protein (eGFP). The expression level of eGFP was slightly higher when using two promoters (Psin2) than using original promoters. The Psin2 promoter was further engineered by modifying the repressor binding site and -35 and -10 regions. Shine-Dalgarno (SD) sequence of the sinI gene was modified to the consensus sequence. Finally, combining the engineered Psin2 promoter with the modified SD sequence increased the expression level of eGFP by about 13.4-fold over the original promoter. Our results suggest that the optimized sinIR promoter could be used as a novel tool for recombinant protein expression in B. subtilis.

• Journal of Microbiology and Biotechnology
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• 제25권7호
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• pp.953-962
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• 2015

Escherichia coli is the most preferred microorganism to express heterologous proteins for therapeutic use, as around 30% of the approved therapeutic proteins are currently being produced using it as a host. Owing to its rapid growth, high yield of the product, costeffectiveness, and easy scale-up process, E. coli is an expression host of choice in the biotechnology industry for large-scale production of proteins, particularly non-glycosylated proteins, for therapeutic use. The availability of various E. coli expression vectors and strains, relatively easy protein folding mechanisms, and bioprocess technologies, makes it very attractive for industrial applications. However, the codon usage in E. coli and the absence of post-translational modifications, such as glycosylation, phosphorylation, and proteolytic processing, limit its use for the production of slightly complex recombinant biopharmaceuticals. Several new technological advancements in the E. coli expression system to meet the biotechnology industry requirements have been made, such as novel engineered strains, genetically modifying E. coli to possess capability to glycosylate heterologous proteins and express complex proteins, including full-length glycosylated antibodies. This review summarizes the recent advancements that may further expand the use of the E. coli expression system to produce more complex and also glycosylated proteins for therapeutic use in the future.

• 대한전기학회논문지:시스템및제어부문D
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• 제55권6호
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• pp.264-273
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• 2006

In this paper, we propose a new architecture of Self-Organizing Fuzzy Polynomial Neural Networks (SOFPNN) by means of consecutive optimization and also discuss its comprehensive design methodology involving mechanisms of genetic optimization. The network is based on a structurally as well as parametrically optimized fuzzy polynomial neurons (FPNs) conducted with the aid of information granulation and genetic algorithms. In structurally identification of FPN, the design procedure applied in the construction of each layer of a SOFPNN deals with its structural optimization involving the selection of preferred nodes (or FPNs) with specific local characteristics and addresses specific aspects of parametric optimization. In addition, the fuzzy rules used in the networks exploit the notion of information granules defined over system's variables and formed through the process of information granulation. That is, we determine the initial location (apexes) of membership functions and initial values of polynomial function being used in the premised and consequence part of the fuzzy rules respectively. This granulation is realized with the aid of the hard c-menas clustering method (HCM). For the parametric identification, we obtained the effective model that the axes of MFs are identified by GA to reflect characteristic of given data. Especially, the genetically dynamic search method is introduced in the identification of parameter. It helps lead to rapidly optimal convergence over a limited region or a boundary condition. To evaluate the performance of the proposed model, the model is experimented with using two time series data(gas furnace process, nonlinear system data, and NOx process data).

• 전기학회논문지
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• 제60권4호
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• pp.862-870
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• 2011

In this paper, we introduce a new topology of Radial Basis Function-based Polynomial Neural Networks (RPNN) that is based on a genetically optimized multi-layer perceptron with Radial Polynomial Neurons (RPNs). This study offers a comprehensive design methodology involving mechanisms of optimization algorithms, especially Fuzzy C-Means (FCM) clustering method and Particle Swarm Optimization (PSO) algorithms. In contrast to the typical architectures encountered in Polynomial Neural Networks (PNNs), our main objective is to develop a design strategy of RPNNs as follows : (a) The architecture of the proposed network consists of Radial Polynomial Neurons (RPNs). In here, the RPN is fully reflective of the structure encountered in numeric data which are granulated with the aid of Fuzzy C-Means (FCM) clustering method. The RPN dwells on the concepts of a collection of radial basis function and the function-based nonlinear (polynomial) processing. (b) The PSO-based design procedure being applied at each layer of RPNN leads to the selection of preferred nodes of the network (RPNs) whose local characteristics (such as the number of input variables, a collection of the specific subset of input variables, the order of the polynomial, and the number of clusters as well as a fuzzification coefficient in the FCM clustering) can be easily adjusted. The performance of the RPNN is quantified through the experimentation where we use a number of modeling benchmarks - NOx emission process data of gas turbine power plant and learning machine data(Automobile Miles Per Gallon Data) already experimented with in fuzzy or neurofuzzy modeling. A comparative analysis reveals that the proposed RPNN exhibits higher accuracy and superb predictive capability in comparison to some previous models available in the literature.

• Journal of Microbiology and Biotechnology
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• 제29권9호
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• pp.1470-1477
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• 2019

${\beta}_2$-adrenergic receptor (${\beta}_2-AR$) was expressed efficiently using Bac-to-Bac Baculovirus Expression System in Sf9 cells as a bio-recognition element for multianalyte screening of ${\beta}$-agonist residues in pork. Sf9 cells were selected as the expression system, and codon optimization of wild-type nucleic acid sequence and time-dependent screening of expression conditions were then carried out for enhancing expression level and biological activity. Under optimum conditions of multiplicity of infection (MOI) = 5 and 48 h post transfection, the protein yield was up to 1.23 mg/ml. After purification by chromatographic techniques, the purified recombinant protein was applied to develop a direct competitive enzyme-linked receptor assay (ELRA) and the efficiency and reliability of the assay was determined. The IC50 values of clenbuterol, salbutamol, and ractopamine were 28.36, 50.70, and $59.57{\mu}g/l$, and clenbuterol showed 47.61% and 55.94% cross-reactivities with ractopamine and salbutamol, respectively. The limit of detection (LOD) was $3.2{\mu}g/l$ and the relevant recoveries in pork samples were in the range of 73.0-91.2%, 69.4-84.6%, and 63.7-80.2%, respectively. The results showed that it had better performance compared with other present nonradioactive receptorbased assays, indicating that the genetically modified ${\beta}_2-AR$ would have great application potential in detection of ${\beta}$-agonist residues.

• 한국양식학회지
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• 제19권3호
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• pp.166-172
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• 2006

동일 종내 미꾸라지(Misgurnus mizolepis) 웅성발생성 인공처녀생식(intraspecific androgenesis)기술을 개발하기 위해 미꾸라지 난 유전물질의 불활성화와 이에 따른 웅성발생성 반수체 유도 조건을 최적화하였다. 다양한 자외선(UV)농도$(0\sim25,650ergs/mm^2)$를 이용하여 미꾸라지 난의 유전학적 불활성화를 시도하였으며 수정률, 부화율 및 반수체 출현율을 평가한 결과 10,800 ergs의 농도에서 가장 우수한 반수체 유도 효율을 보였으며 최초 처리 난 중 50%이상의 웅성발생성 반수체 수율을 확보할 수 있었다. 웅성발생성 개체는 전형적인 haploid syndrome의 특징을 나타내었고 flow cytometry분석 결과 정확히 미꾸라지 반수체(1.4 pg/cell)의 DNA함량을 보유하는 것으로 나타났다. 인위적으로 삽입된 형질전환 유전자 표지를 이용하여 부계 특이적인 인공처녀생식 여부를 확인하였으나 일부 반수체자어들에서$(8\sim11%)$ 모계 유전물질의 잔류가 검출되었다.

• Molecules and Cells
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• 제43권11호
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• pp.909-920
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• 2020

Cytosolic Ca²⁺ levels ([Ca²⁺]_c) change dynamically in response to inducers, repressors, and physiological conditions, and aberrant [Ca²⁺]_c concentration regulation is associated with cancer, heart failure, and diabetes. Therefore, [Ca²⁺]_c is considered as a good indicator of physiological and pathological cellular responses, and is a crucial biomarker for drug discovery. A genetically encoded calcium indicator (GECI) was recently developed to measure [Ca²⁺]_c in single cells and animal models. GECI have some advantages over chemically synthesized indicators, although they also have some drawbacks such as poor signal-to-noise ratio (SNR), low positive signal, delayed response, artifactual responses due to protein overexpression, and expensive detection equipment. Here, we developed an indicator based on interactions between Ca²⁺-loaded calmodulin and target proteins, and generated an innovative GECI sensor using split nano-luciferase (Nluc) fragments to detect changes in [Ca²⁺]_c. Stimulation-dependent luciferase activities were optimized by combining large and small subunits of Nluc binary technology (NanoBiT, LgBiT:SmBiT) fusion proteins and regulating the receptor expression levels. We constructed the binary [Ca²⁺]_c sensors using a multicistronic expression system in a single vector linked via the internal ribosome entry site (IRES), and examined the detection efficiencies. Promoter optimization studies indicated that promoter-dependent protein expression levels were crucial to optimize SNR and sensitivity. This novel [Ca²⁺]_c assay has high SNR and sensitivity, is easy to use, suitable for high-throughput assays, and may be useful to detect [Ca²⁺]_c in single cells and animal models.

• 분석과학
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• 제16권5호
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• pp.349-357
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• 2003

유전학적으로 밝혀지지 않은 생물학적 연구에 있어서 작은 분자량의 아미노산 또는 홀몬과 같은 대사체들은 그 변화와 여러 생물학적 데이타와 결합 되어 직접적인 생화학적 의미 해석을 가능하게 한다. 미량의 생체 시료에 존재하는 아미노산을 분석하기 위해서 HPLC/FLD를 사용하였으며, 감도가 우수하고 반응시간이 빠른 유도체화 방법인 OPA/3-MPA로 형광 유도체화 하여 세포 배지를 바탕시료로 하여 아미노산을 분석하였다. 유도체화물의 시간에 대한 불안정성을 개선하기 위하여 다 단계의 injector program을 사용하여 유도체화 반응 후 시료 주입시간을 일정하게 조절하여 유도체화 과정 중 발생할 수 있는 불순물 제거 및 정량성을 개선하였다. 19종 아미노산의 표준 검정 곡선은 0.5 - 100.0 ppb의 범위에서 $r^2=0.99$ 이상의 직선성을 나타냈으며, 검출 한계는 1.70 pmol(GLU) - 23.81 pmol(SER) 범위로 측정되었다. 이를 통해 다량의 세포를 대상으로 하는 대사 프로필을 위해 감도가 우수하고 안정적으로 정량할 수 있는 분석법을 설정하였다.

• 한국수정란이식학회지
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• 제30권1호
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• pp.45-50
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• 2015

본 연구는 백한우 개체 1두에서 성판별된 정자를 제조하고 원하는 성을 가진 후대를 생산하고자 인공수정을 실시하였다. 채정이 가능한 백한우 종모우 1두를 선발하고, 정자 성 분리 전용 유세포 분리기인 MoFlo XDP기기로 성분리 동결정액을 생산하였다. 성분리 X 정자의 농도에 따른 임신율과 출산율을 비교하기 위하여 대조군으로서 KPN 768정액을 이용하였으며 그 총 정자수는 $20{\times}10^6$로 추정되었다. 실험군으로서 성 판별된 백한우 X 정자를 이용하였으며, 정자수 $2{\times}10^6/straw$와 $4{\times}10^6/straw$를 처녀우 한우에 2회 인공수정하였다. 백한우의 정액 기형도는 $24.9{\pm}7.31%$로 관찰되었으며, 중편부 반전 정자기형이 약간 높은 정자로 검사되었다(11.7%). 성판별된 백한우 X-정자수가 $2{\times}10^6/straw$와 $4{\times}10^6/straw$를 인공 수정할 때 실험군에서 임신율의 차이는 없었으며, 보증씨수소 정자를 이용한 대조군의 경우, 임신율이 유의적으로 높게 나타났다(p<0.05). 대조군 KPN 768와 실험군 $2{\times}10^6/straw$ 정자와 $4{\times}10^6/straw$ 정자 처리군에서 임신율은 각각 85.0%, 26.3% 및 50 %로 관찰되었다. 산자 생산율은 각각 80.0%, 15.8% 및 21.4% 로 관찰되었고, 암컷 출현율은 각각 43.8%, 100% 및 100%로 조사되었다. 이러한 결과는 백한우 성 판별된 정자로 원하는 성을 가진 후대 생산이 가능함을 보여주고 있으며, 성 판별된 정자를 활용하여 원하는 성을 가진 백한우 후대를 생산하는데 최적화된 정자수의 대한 기본 자료를 제공할 수 있을 것으로 기대된다.