• Biotechnology and Bioprocess Engineering:BBE
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• 제7권1호
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• pp.1-5
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• 2002

A fusion protein, consisting of a human epidermal growth factor (hEGF) as the recognition domain and human angiogenin as the toxin domain, can be used as a targeted therapeutic against breast cancer cells among others. The fusion protein was expressed as inclusion body in recombinant E. coli, and when the conventional, solution-phase refolding process was used the refolding yield was very low due to severe aggregation. It was probably because of the opposite electric charge at a neutral pH resulting from the vastly different pI values of each domain. The solid-phase refolding process that exploited the ionic interactions between ionic exchanger surface and the fusion protein was tried, but the adsorption yield was also very low, below $ 30\%$, regardless of the resins and pH conditions used. Therefore, to provide a higher ionic affinity toward the solid matrix, six lysine residues were tagged to the N-terminus of the hEGF domain. When heparin-Sepharose was used as the matrix, the adsorption capacity increased 2.5-3 times to about $88\%$. Besides the intrinsic affinity of angiogenin to heparin, the poly-lysine tag provided additional ionic affinity. And the subsequent refolding yield increased nearly 13-fold, from ca. $4.8\%$ in the conventional refolding of the untagged fusion protein to $63.6\%$. The process was highly reproducible. The refolded protein in the column eluate retained RNase bioactivity of angiogenin.

• Genomics & Informatics
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• 제21권1호
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• pp.9.1-9.13
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• 2023

Matrix metalloproteinase-9 (MMP-9) is a zinc and calcium-dependent proteolytic enzyme involved in extracellular matrix degradation. Overexpression of MMP-9 has been confirmed in several disorders, including cancers, Alzheimer's disease, autoimmune diseases, cardiovascular diseases, and dental caries. Therefore, MMP-9 inhibition is recommended as a therapeutic strategy for combating various diseases. Cinnamic acid derivatives have shown therapeutic effects in different cancers, Alzheimer's disease, cardiovascular diseases, and dental caries. A computational drug discovery approach was performed to evaluate the binding affinity of selected cinnamic acid derivatives to the MMP-9 active site. The stability of docked poses for top-ranked compounds was also examined. Twelve herbal cinnamic acid derivatives were tested for possible MMP-9 inhibition using the AutoDock 4.0 tool. The stability of the docked poses for the most potent MMP-9 inhibitors was assessed by molecular dynamics (MD) in 10 nanosecond simulations. Interactions between the best MMP-9 inhibitors in this study and residues incorporated in the MMP-9 active site were studied before and after MD simulations. Cynarin, chlorogenic acid, and rosmarinic acid revealed a considerable binding affinity to the MMP-9 catalytic domain (ΔG_binding < -10 kcal/ mol). The inhibition constant value for cynarin and chlorogenic acid were calculated at the picomolar scale and assigned as the most potent MMP-9 inhibitor from the cinnamic acid derivatives. The root-mean-square deviations for cynarin and chlorogenic acid were below 2 Å in the 10 ns simulation. Cynarin, chlorogenic acid, and rosmarinic acid might be considered drug candidates for MMP-9 inhibition.

• 대한약학회:학술대회논문집
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• 대한약학회 2003년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.1
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• pp.290.3-291
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• 2003

To increase detection sensitivity for multi-DDT residues (o,p-/p,p-DDT, o,p-/p,p-DDE, o,o-/o,p-DDD) analysis, a highly selective sample clean-up method was introduced prior to GC/MS analysis using immunoaffinity column. The immunoaffinity matrix was prepared by coupling IgG fraction of DDT antiserum to cyanogens bromide activated Sepharose 4B. Three DDT antisera (DDA-1, DDHP-2, DDCP-3) were test for affinity column ligand that obtained by imunizing respective DDT immunogen to rabbits, and IgG was purified using protein A affinity purification. (omitted)

• KSBB Journal
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• 제21권4호
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• pp.279-285
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• 2006

본 연구에서는 trypsin을 모델 단백질로 하여 단백질 본연의 환성을 유지할 수 있는 고정화 방법을 찾기 위하여 공유결합방법과 친화력 결합방법을 이용하여 trypsin을 고정화 하였다. Streptavidin-biotin system을 이용한 고정화 방법은 bioactivity 유지측면에서 공유결합 방법보다 우수함을 확인하였다. 하지만 streptavidin-biotin system을 이용하였을 때 고정화 수율이 낮은 것은 해결해야 할 과제이다. 분자량이 다른 기질들(BAPNA, insulin, BSA)을 대상으로 고정화 trypsin의 부위 특이적 절단 특성을 분석한 결과 streptavidin-biotin에 의해 고정화된 trypsin이 절단효율도 높고 sequence coverage도 높은 것으로 확인되었다. 또한 공유결합된 trypsin은 견고한 분자구조를 나타낸 반면 streptavidin-biotin system으로 고정화된 trypsin은 유연성이 높은 것을 QCM-D를 이용하여 관찰할 수 있었다. 따라서 streptavidin-biotin system에 의한 고정화 방법에서 streptavidin-biotin 결합이 일종의 spacer arm 역할을 하면서 고정화된 trypsin의 분자유연성을 향상시켜 절단반응의 부위특이성과 절단수율을 향상시키는 것으로 판단되었다.

• Journal of Microbiology and Biotechnology
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• 제12권2호
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• pp.196-203
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• 2002

A novel process for urokinase purification was studied using p-aminobenzamidine as the ligand and sepharose 4B as the matrix. The adsorption, washing, and elution conditions were optimized by an unusual method. An adsorption buffer containing 2.5 M NaCl and $1\%$ Tween 80 facilitated the adsorption of urokinase on the affinity media and prevented contaminants from binding to the p-aminobenzamidine affinity gel. It was found that $5\%$ Tween 80 removed most of the contaminants from the affinity column. A 0.2 M glycine elution buffer containing 0.5 M NaCl (pH 3.0) was found to have a strong elution ability with a high recovery and purity of urokinase. A crude urokinase material of231 IU/mg protein from human urine was purified to 124,300 IU/mg protein with a purification factor of 538 and yield of $86.7\%$. As a result, a high purity urokinase was obtained with only a single affinity chromatography step. The purification process was successfully scaled-up to a 2-1 chromatography column. The resulting urokinase eluate could be directly lyophilized, thereby complying with Chinese pharmacopoeia (1995 version) standards.

• 한국패류학회지
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• 제3권1호
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• pp.3-14
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• 1987

The electrophoretic and innunological cnalyses of organic matrices in the shells of freshwater bivalves were made in order to dlucidate the biochemical characteristics and species-specific differinces of the applied shells, The water-soluble and insoluble matrices of four species of freshwater bivalves, Andodonta fukudai, Unio douglasiae, Lanceolaria and Chrbicula fluminea, were used as analytical materials, There was non-identity in immuno affinity between anti soluble matrix(anti-Sm) and anti insoluble matrix(anti-ISM)sera against the organic matrix of Andodnta fukudai. The SMs of four species (S. fukudai, U.douglasiae, L. acrorhyncha, and C. fluminea) showed the differences in the precipitate arcs at the level of family, though ISMs did mot show differences. In the electrophoretic analysis, all foru species had two SDS-electrophoretic bands of SM, of which molecular wights appeared to be lower than 55,000, shereas the native organic matrices of foru speceis had higher molecular weighrs than those from SDS-dldctrophoresis. Only calcium ion among many ions in extrapallial fluid(EPF) caused SM to change into insoluble molecules, thus the EPF pretreated with Ca++did not form the precipitate arc when did the immuno diffusion whth anti SM serum. ISM precursor may be polymerized into macromolecules like periostracin, a precusor of periostracum, judging from the similat polymerization patterns in 0.1M Tris formate buffer(pH 3); they may be made insoluble macromolecules due to their strong natrue of hydrophobicity.

• 한국항공우주학회지
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• 제38권5호
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• pp.467-476
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• 2010

본 연구에서는 개념 설계 과정에서 항공 인증 규정 및 의사결정모델을 이용하여 최적화 문제 구성과 항공기 기준형상을 선정하는 프로세스를 정립하고, 이를 소형제트항공기에 적용하였다. 항공기 안전성을 보장하기 위한 최소안전 요구조건인 항공 인증 규정은 항공기 설계 초기단계에서부터 고려되어야 하는 사항으로 반드시 만족해야 한다. 인증 규정 및 사용자 요구도 분석 후, 산업공학기법인 Affinity Diagram, Nested Column Diagram, Quality Function Deployment (QFD), Pugh Concept Selection Matrix와 같은 의사결정모델을 사용하여 alternative 형상군에 대한 평가를 수행하였다. 그 후 Best alternative 형상에 대한 설계가능영역 분석을 통해 항공 인증 규정에 적합하고 객관적인 문제 구성 및 항공기 기준형상을 도출 할 수 있었다.

• 한국환경농학회지
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• 제25권4호
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• pp.371-381
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• 2006

New proteomic techniques have been pioneered extensively in recent years, enabling the high-throughput and systematic analyses of cellular proteins in combination with bioinformatic tools. Furthermore, the development of such novel proteomic techniques facilitates the elucidation of the functions of proteins under stress or disease conditions, resulting in the discovery of biomarkers for responses to environmental stimuli. The ultimate objective of proteomics is targeted toward the entire proteome of life, subcellular localization biochemical activities, and the regulation thereof. Comprehensive analysis strategies of proteomics can be classified into three categories: (i) protein separation via 2-dimensional gel electrophoresis (2-DE) or liquid chromatography (LC), (ii) protein identification via either Edman sequencing or mass spectrometry (MS), and (iii) proteome quantitation. Currently, MS-based proteomics techniques have shifted from qualitative proteome analysis via 2-DE or 2D-LC coupled with off-line matrix assisted laser desorption ionization (MALDI) and on-line electrospray ionization (ESI) MS, respectively, toward quantitative proteome analysis. In vitro quantitative proteomic techniques include differential gel electrophoresis with fluorescence dyes. protein-labeling tagging with isotope-coded affinity tags, and peptide-labeling tagging with isobaric tags for relative and absolute quantitation. In addition, stable isotope-labeled amino acids can be in vivo labeled into live culture cells via metabolic incorporation. MS-based proteomics techniques extend to the detection of the phosphopeptide mapping of biologically crucial proteins, which ale associated with post-translational modification. These complementary proteomic techniques contribute to our current understanding of the manner in which life responds to differing environment.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2001년도 추계학술발표대회
• /
• pp.197-203
• /
• 2001

A fusion protein, consisting of human epidermal growth factor as a recognition domain and human angiogenin as a toxin domain, can be used as a targeted therapeutic against breast cancer cells among others. The fusion protein was expressed as inclusion body in recombinant E. coli, and when the conventional, solution-phase refolding process was used the refolding yield was very low due to severe aggregation, probably due to the opposite surface charge due to vastly different pI values of each domain. Solid-phase refolding process exploiting ionic interactions between the solid matrix and the protein was tried, but the ionic binding yield was very low regardless of the resins and pH conditions used. To provide higher affinity toward the solid matrix, six lysine residues were tagged to the N -terminus of the hEGF domain When the cation exchange resins such as heparin- or CM-Sepharose were used as the matrix, the adsorption capacity increased 2.5-3 times and the subsequent refolding yield increased nearly IS times compared to the conventional process.

• ETRI Journal
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• 제35권2호
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• pp.311-320
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• 2013

In most spectral clustering approaches, the Gaussian kernel-based similarity measure is used to construct the affinity matrix. However, such a similarity measure does not work well on a dataset with a nonlinear and elongated structure. In this paper, we present a new similarity measure to deal with the nonlinearity issue. The maximum flow between data points is computed as the new similarity, which can satisfy the requirement for similarity in the clustering method. Additionally, the new similarity carries the global and local relations between data. We apply it to spectral clustering and compare the proposed similarity measure with other state-of-the-art methods on both synthetic and real-world data. The experiment results show the superiority of the new similarity: 1) The max-flow-based similarity measure can significantly improve the performance of spectral clustering; 2) It is robust and not sensitive to the parameters.