• 자연과학논문집
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• 제9권1호
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• pp.25-29
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• 1997

Glucocorticoid에 의한 항염증 작용의 second messenger로 생각되어지는 annexin superfamily중 하나인 37 kDa의 단백질, lipocortin-1의 작용기작을 이해할 목적으로 in vivo에서 protein-protein interaction을 인식하는 yeast-based genetic assay인 yeast two assay를 통하여 lipocortin-1과 결합하는 단백질 유전자를 분리하여 조사하였다. 이 방법으로 실험을 수행한 결과 분리된 유전자가 human serine proteinase 유전자와 homology가 있는 것으로 밝혀졌다.

• 대한수의학회지
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• 제30권2호
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• pp.187-192
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• 1990

돼지로부터 분리한 Staphylococcus hyicus subsp hyicus 489주의 protein A 존재여부와 함량을 indirect hemagglutination 및 enzyme-linked immunosorbent assay(ELISA)법으로 조사하였다. Indirect hemagglutination text에 의하여 cell-bound protein A 및 extracellular protein A 보유균은 489주 중 각각 87.7% 및 36.0%로 나타났다. ELISA법에 의한 이들 균의 protein A함량 측정에서 전균주의 extracellular protein A는 1ng/ml미만이었으며, cell-bound protein A함량은 대부분의 균주에서 1ng/ml 미만이었고 11주가 25~108ng/ml 수준이었다.

• BMB Reports
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• 제51권10호
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• pp.526-531
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• 2018

Protein-Protein Interactions (PPIs) play essential roles in diverse biological processes and their misregulations are associated with a wide range of diseases. Especially, the growing attention to PPIs as a new class of therapeutic target is increasing the need for an efficient method of cell-based PPI analysis. Thus, we newly developed a robust PPI assay (SeePPI) based on the co-translocation of interacting proteins to the discrete subcellular compartment 'processing body' (p-body) inside living cells, enabling a facile analysis of PPI by the enriched fluorescent signal. The feasibility and strength of SeePPI (${\underline{S}}ignal$ ${\underline{e}}nhancement$ ${\underline{e}}xclusively$ on ${\underline{P}}-body$ for ${\underline{P}}rotein-protein$ ${\underline{I}}nteraction$) assay was firmly demonstrated with FKBP12/FRB interaction induced by rapamycin within seconds in real-time analysis of living cells, indicating its recapitulation of physiological PPI dynamics. In addition, we applied p53/MDM2 interaction and its dissociation by Nutlin-3 to SeePPI assay and further confirmed that SeePPI was quantitative and well reflected the endogenous PPI. Our SeePPI assay will provide another useful tool to achieve an efficient analysis of PPIs and their modulators in cells.

• 약학회지
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• 제50권4호
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• pp.268-271
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• 2006

Protein kinase UL97 is an unusual protein kinase that can phosphorylate nucleoside analogs as well as protein/peptide. Previously we found a H2B-derived peptide, KESYSVYVYKV and reported that the P+5 position (K) is important. To further understand the substrate specificity at the P+5 position, we introduced spot assay system and showed that a peptide containing K residue among other amino acids at the P+5 position is the best substrate. Also other residues such as M, I, L, or G are good enough to be substrate of UL97. This result may aid the discovery of a new antiviral inhibitor.

• Journal of Microbiology and Biotechnology
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• 제12권2호
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• pp.286-291
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• 2002

Histone deacetylase I (HDAC1) works as one of the components in a nucleosome remodeling (NuRD) complex that consists of several proteins, including metastasis-associated protein 1 (MTA1). Since the protein-protein interaction of HDAC1 and MTA1 would appear to be important for both the integrity and functionality of the HDAC1 complex, the interruption of the HDAC1 and MTA1 interaction may be an efficient way to regulate the biological function of the HDAC1 complex. Based on this idea, a yeast two-hybrid system was constructed with HDAC1 and MTA1 expressing vectors in the DNA binding and activation domains, respectively. To verify the efficiency of the assay system, 3,500 microbial metabolite libraries were tested using the paper disc method, and KB0699 was found to inhibit the HDAC1 and MTA1 interaction without any toxicity to the wild-type yeast. Furthermore, KB0699 blocked the interaction of HDAC1 and MTA1 in an in vitro GST pull down assay and induced morphological changes in B16/BL6 melanoma cells, indicating the interruption of the HDAC1 complex function. Accordingly, these results demonstrated that the yeast assay strain developed in this study could be a valuable tool for the isolation of a HDAC1 complex disruptor.

• Journal of Microbiology and Biotechnology
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• 제16권9호
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• pp.1384-1391
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• 2006

The signal transduction system is one of the most important devices involved in maintaining life, and many protein kinases are included in the cellular signal transduction system. Finding a protein kinase inhibitor is very valuable, as it can be used to study cell biology and applied to pharmaceuticals. For the efficient and rapid screening of protein kinase inhibitors, two assay systems were combined; the nonradioactive protein kinase assay system that uses an FITC-labeled IRS-2 peptide and the cell-based paper disc assay system that uses Streptomyces griseus as the indicator strain. Among 330 kinds of herb extracts tested, the extract of Dalbergia odorifera exhibited the strongest inhibitory activity in the two assay systems and was selected for further isolation. Based on solvent extraction and many steps of chromatography, seven compounds were finally separated to homogeneity and their structures determined by $^{1}H$ and $^{13}C$ NMR spectroscopies. Four were to be flavonoids and identified as butin ($C_{15}H_{12}O_5$, Mw=272.07), 3'-hydroxymelanetin ($C_{16}H_{12}O_6$, Mw=300.06), liquiritigenin ($C_{15}H_{12}O_4$, Mw=256.07), and 2'-hydroxyformononetin ($C_{16}H_{12}O_{5}$, Mw=284.07). 3'-Hydroxymelanetin inhibited the phosphorylation of the GSK3 protein by Akt to 37% at a concentration of $10{\mu}g/ml$ and showed the strongest cytotoxicity ($ED_{50}<50{\mu}g/ml$) against the human cancer cell line HCT116. Under the same conditions, liquiritigenin also inhibited the phosphorylation of GSK3 by Akt to 26%, and its cytotoxicity against the HCT116 cell line was lower than $100{\mu}g/ml$.

• 한국안광학회지
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• 제19권1호
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• pp.51-57
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• 2014

목적: 본 연구에서는 콘택트렌즈 단백질제거제 개발 시 요구되는 단백질 제거효능의 적절한 평가를 위한 시험법을 확립하고 이를 실제 콘택트렌즈에 침착된 단백질의 제거효율을 측정한 결과와 비교, 분석하여 단백질제거제의 효능 평가법으로 제시하고자 하였다. 방법: 대한약전에 제시된 단백소화력 시험법을 이용하여 파파인, 판크레아틴, 섭틸리신 A 및 프로테아제와 각각의 효소가 포함된 단백질제거 정 또는 용액의 효능평가에 적절한 시험조건을 찾고자 하였다. 또한, balafilcon A 재질 렌즈에 인위적으로 침착시킨 단백질을 시판되고 있는 단백질제거 정 또는 용액으로 제거하여 세척효율을 확인하고 이들 방법의 상관관계를 분석하였다. 결과: 판크레아틴과 판크레아틴 함유 제품의 경우 단백소화력 시험법으로 평가하였을 때 둘 다 약전에서 제시하는 판크레아틴 단백소화력 기준값인 28 IU/mg를 충족하였다. 프로테아제와 삼염화아세트산 B 용액으로 실험한 섭틸리신 A의 경우는 단백소화력 시험법으로 평가하였을 때 제조사에서 제시한 효소활성 값을 충족하였으나, 파파인과 삼염화아세트산 A 용액으로 실험한 섭틸리신 A는 제조사에서 제시된 효소활성 값에 해당하는 단백소화력이 측정되지 않았다. 시판되는 단백질제거제의 경우 판크레아틴을 함유한 제품을 제외한 나머지 세 가지 제품은 단백소화력 시험법으로는 제조사에서 명시한 효소들의 단백질 효소활성 값을 확인할 수 없었다. 그러나 실제 렌즈에 침착된 단백질의 제거정도를 측정하였을 때에는 파파인을 함유한 제품을 제외한 3종의 단백질제거제는 모두 90%가 넘는 단백질 제거효율을 보였다. 파파인 함유 단백질제거제의 경우 단백소화력 시험법으로는 효능 측정이 불가능하였으나 실제 렌즈에 침착된 누액단백질 제거효율은 73.72%에 이르렀다. 결론: 본 연구결과로 콘택트렌즈 단백질제거제의 효능은 함유되어 있는 단백분해효소의 종류에 따라 시험법을 달리하여 평가되어야 함을 확인할 수 있었다. 즉, 판크레아틴, 프로테아제, 섭틸리신 A를 함유하는 단백질제거제는 단백소화력 시험법과 단백질제거효율 측정법으로 효능평가가 가능하고, 파파인을 함유하는 단백질제거제의 평가는 콘택트렌즈를 이용한 단백질제거효율 측정법만이 효율적임을 제안할 수 있다.

• 한국식품영양과학회지
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• 제14권2호
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• pp.202-213
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• 1985

The protein nutritional quality of foods has become an important factor to food processors with the advent of nutritional labeling regulations for foods. Then, as is true today, the officially approved assay for protein nutritional quality was the rat based protein efficiency ratio(PER) bioassay. The PER bioassay requires a minimum of 28 days to performe, and is therefore not applicable to routine quality assurance use by the food industry. Within the past ten years there has been a research emphasis placed on the development of rapid, inexpensive, biological and/or chemical based assays for protein nutritional quality. It was hoped that if a rapid assay could be developed and thoroughly tested, it could be used in lieu of the PER bioassay in the day-to-day quality assurance screening of food ingredients and products. The rapid assays developed in the hope of attaining this goal have been based on microorganisms, proteolytic enzymes, and amino acid profiles, as well as combinations of the above. In this review, it will be described and briefly discussed many of procedures which had contributed conceptually as well as practically to the development of in vitro methods for the evaluation of protein quality. Special emphasis will be placed on the C-PER(computed protein efficiency ratio) assay which combines data from in vitro protease digestion and amino acid composition to predict protein nutritional quality designed by Satterlee et al. (1980), and the DC-PER(discriminant computed PER) which is a method of estimating protein quality based on rat assay and in vitro digestibility obtained using solely essential amino acid data will be also introduced.

• Animal cells and systems
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• 제6권4호
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• pp.311-315
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• 2002

The RAD4 gene is essential for nucleotide excision repair in Saccharomyces cerevisiae. It has been known that the deduced amino acid sequence of Rad4 protein contains three DNA-dependent ATPase/helicase motifs. To determine the biochemical activities and functional role of RAD4 the Rad4 protein was expressed and purified. Immunoblot analysis showed a specific band of 21 kDa, which was well-matched with the size of open reading frame of the RAD4 gene. The purified Rad4 protein had no detectable helicase activity. However, the protein could interact with double stranded oligonucleotides, as judged by mobility shift assay. This result suggests that the Rad4 protein is a DNA binding protein.

• 약학회지
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• 제37권6호
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• pp.598-604
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• 1993

The calyx extract of Campsis grandiflora displayed inhibitory activity against protein kinase C from the bovine brain. Separation guided by protein kinase C enzyme assay and bleb forming assay led to isolation of a potent protein kinase C inhibitor that was identified as a known phenylpropanoid glycoside, verbascoside. It suppressed completely bleb-formation of K562 cell surface induced by phorbol 12,13-dibutylate at the concentration of 60 $\mu\textrm{g}$/ml and IC$_{50}$ of the protein kinase C occured at 20 $\mu{M}$. This compound was tested for cytotoxic activity against ten human tumor cell lines in vitro. it exhibited moderate cytotoxic activity against skin tumor cell line M14 (IC$_{50}$ 2.2 $\mu\textrm{g}$/ml) and very weak cytotoxicity against other cell lines (IC$_{50}$>10 $\mu\textrm{g}$/ml)