• Journal of Microbiology and Biotechnology
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• 제6권6호
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• pp.456-460
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• 1996

Recombinant Pseudomonas lipase was used to study protein aggregation and adsorption upon in vitro refolding. Protein adsorption as well as aggregation was responsible for major side reactions upon in vitro refolding as a function of protein concentration. The optimal range of protein concentration was determined by the relative contribution of protein aggregation and adsorption. Above the optimal range, the yield of active lipase inversely correlated with protein aggregation, showing a competition between folding and aggregation. However, adsorption of protein rather than protein aggregation is thought to contribute as a major side reaction of the refolding process at sub-optimal concentrations at which the formation of aggregates should be more reduced. Protein aggregation was influenced by the amount of guanidine hydrochloride in the refolding solvent. The refolding temperature was a critical factor determining the extent of protein aggregation. The refolding yield was also affected by the dilution fold and dilution mode, which suggests that the refolding process might kinetically compete with the rate of mixing.

• EDISON SW 활용 경진대회 논문집
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• 제4회(2015년)
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• pp.70-75
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• 2015

Fasciclin 1 (FAS1) is an extracellular protein whose aggregation in cornea leads to visual impairment. While a number of FAS1 mutants have been studied that exhibit enhanced/decreased aggregation propensity, no structural information has been provided so far that is associated with distinct aggregation potential. In this study, we have investigated the structural and thermodynamic characteristics of the wild-type FAS1 and its two mutants, R555Q and R555W, by using molecular dynamics simulations and three-dimensional reference interaction site model (3D-RISM) theory. We find that the hydrophobic solvent accessible surface area increases due to hydrophobic core repacking in the C-terminus caused by the mutation. We also find that the solvation free energy of the mutants increases due to the enhanced non-native H-bonding. These structural and thermodynamic changes upon mutation contribute to understand the aggregation of these mutants.

• Journal of Pharmaceutical Investigation
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• 제36권2호
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• pp.115-121
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• 2006

The aim of this study was to develop new protein/peptide depot system instead of poly(DL-lactic acid-coglycolic acid) (PLGA) microspheres. Pullulan was chemically modified by the addition of acetic anhydride (pullulan acetate; PA) and then investigated as new depot system for protein/peptide delivery. PA microspheres (PAM) with lysozyme as a model protein were prepared by w/o/w double emulsion method. The microspheres had a mean size of 10-50 mm with a spherical shape. The size distributions reduced with increasing the degree of acetylation. The loading efficiency of lysozyme was also increased. Lysozyme aggregation behavior in the microsphere was monitored to estimate the change of protein stability during preparation step. The ratios of protein aggregation in PAMs are lower than that of PLGA microsphere, in particular, PA 5 showed lowest as about 16%. The result indicated that the increase of acetylation suppressed the aggregation of protein. The release profiles of lysozyme from PAMs were significantly different. High acetylation effectively improved lysozyme release kinetics by reducing initial burst release and extending continuous release over a period of time. To check the effect of preservation for structural stability of lysozyme, the activity of lysozyme released from PA 5 was also observed. The activity of lysozyme was maintained almost 100% for 25 day. Therefore, PAM may become to a useful carrier for delivery of protein/peptide drugs, if it will be supported by biocompatibility and biodegradability results.

• 한국자기공명학회논문지
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• 제24권3호
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• pp.91-95
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• 2020

Transthyretin (TTR) is an abundant protein in blood plasma and cerebrospinal fluid (CSF), working as a homo-tetrameric complex to transport thyroxine (T₄) and a holo-retinol binding protein. TTR is well-known for its amyloidogenic property; several types of systemic amyloidosis diseases are caused by aggregation of either wild-type TTR or its variants, for which more than 100 mutations were reported to increase the amyloidogenicity of TTR. The rate-limiting step of TTR aggregation is the dissociation of a monomeric subunit from a tetrameric complex. A wide range of biochemical and biophysical techniques have been employed to elucidate the TTR aggregation processes, among which nuclear magnetic resonance (NMR) spectroscopy contributed much to characterize the structural and functional features of TTR during its aggregation processes. The present review focuses on discussing the recent advances of our understanding to the amyloidosis mechanism of TTR and to the structural features of its monomeric aggregation-prone state in solution. We expect that the present review provides novel insights to appreciate the molecular basis of TTR amyloidosis and to develop novel therapeutic strategies to treat diverse TTR-related diseases.

• Journal of Applied Biological Chemistry
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• 제65권3호
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• pp.167-172
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• 2022

Pathophysiological reaction of platelets in the blood vessel is an indispensable part of thrombosis and cardiovascular disease, which is the most common cause of death in the world. In this study, we performed in vitro assays to evaluate antiplatelet activity of artemether in human platelets and attempted to identify the mechanism responsible for protein phosphorylation. Artemether is a derivative of artemisinin, known as an active ingredient of Artemisia annua, which has been reported to be effective in treating malaria, and is known to function through antioxidant and metabolic enzyme inhibition. However, the role of artemether in platelet activation and aggregation and the mechanism of action of artemether in collagen-induced human platelets are not known until now. In this study, the effect of artesunate on collagen-induced human platelet aggregation was confirmed and the mechanism of action of artemether was clarified. Artemether inhibited the phosphorylation of PI3K/Akt and Mitogen-activated protein kinases, which are phosphoproteins that are known to act in the signal transduction process when platelets are activated. In addition, artemether decreased TXA₂ production and decreased granule secretion in platelets such as ATP and serotonin release. As a result, artemether strongly inhibited platelet aggregation induced by collagen, a strong aggregation inducer secreted from vascular endothelial cells, with an IC₅₀ of 157.92 μM. These results suggest that artemether has value as an effective antithrombotic agent for inhibiting the activation and aggregation of human platelets through vascular injury.

• BMB Reports
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• 제35권6호
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• pp.590-594
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• 2002

To elucidate the effect of gamma-irradiation on the molecular properties of myoglobin, the secondary and tertiary structures, as well as the molecular weight size of the protein, were examined after irradiation at various irradiation doses. Gamma-irradiation of myoglobin solutions caused the disruption of the ordered structure of the protein molecules, as well as degradation, cross-linking, and aggregation of the polypeptide chains. A SDS-PAGE study indicated that irradiation caused initial fragmentation of the proteins and subsequent aggregation, due to cross-linking of the protein molecules. The effect of irradiation on the protein was more significant at lower protein concentrations. Ascorbic acid protected against the degradation and aggregation of proteins by scavenging oxygen radicals that are produced by irradiation. A circular dichroism study showed that an increase of the irradiation decreased the a-helical content of myoglobin with a concurrent increase of the aperiodic structure content. Fluorescence spectroscopy indicated that irradiation increased the emission intensity that was excited at 280 nm.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 2001년도 Proceedings of 2001 International Symposium
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• pp.146-148
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• 2001

The objective of this study was to investigate the behavior of ribonuclease A (RNase) at the water/methylene chloride interface. It was aimed at better understanding the denaturation of proteins upon emulsification. RNase was vulnerable to the interface-induced aggregation reactions that led to formation of water-insoluble aggregates upon emulsification. Biochemical analyses demonstrated that intermolecular covalent linkages might have been involved in the aggregation reactions. The protein instability observed with emulsification was traced to consequences of protein adsorption and conformational rearrangements at the interface. These results indicated that emulsifying aqueous protein solutions in organic solvents should be handled with care, since emulsification could bring denaturation and aggregation to proteins.

• 한국식품저장유통학회:학술대회논문집
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• 한국식품저장유통학회 2003년도 제23차 추계총회 및 국제학술심포지움
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• pp.135.1-135
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• 2003

To elucidate the effect of gamma-irradiation on the molecular properties of hemoglobin, the secondary, tertiary structure, and the molecular weight size of the protein were examined after irradiation at 0.5, 1, 5, and 10 kGy. Gamma-irradiation of hemoglobin solutions caused the disruption of the ordered structure of the protein molecules, as well as degradation, cross-linking, and aggregation of the polypeptide chains. A SDS-PAGE study indicated that irradiation caused initial fragmentation of the proteins and subsequent aggregation due to cross-linking of the protein molecules. The effect of irradiation on the protein was more significant at lower protein concentrations. Ascorbic acid decreased the degradation and aggregation of proteins by scavenging oxygen radicals that were produced by irradiation. A circular dichroism study showed that irradiation decreased the helical content of hemoglobin with a concurrent increase of the aperiodic structure content. Fluorescence spectroscopy indicated that irradiation decreased the emission intensity that was excited at 280 nm.

• 생명과학회지
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• 제32권9호
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• pp.729-733
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• 2022

수용성 단백질이 비정상적인 불용성 응집(aggregate)으로 전환되면 질환 등 여러 문제를 야기된다. 대장균 트립토판 중합효소 α 소단위체(αTS)는 가장 흔한 구조의 하나인 TIM 배럴 구조를 가지고 있다. 이전의 연구에서 불용성 응집이 일어나는 여러 잔기치환체(Y4C, S33L, P28L, P28S, G44S, D46N, P96L, P96S)를 얻었다. 본 연구에서는 높은 안정성과 도메인 협동성 성질을 보여주는 Y173F가 다른 잔기자리에 치환으로 유도된 응집 형성을 억제할 수 있는 지 여부를 조사했다. 8개 모두에서 억제효과를 보여 주었다. 단백질 분리가 가능한 P28L αTS를 분석한 결과, 이차구조함량의 감소, 안정성 감소, 소수성표면 증가 등의 구조변화특성을 보여주었다. Y173F가 첨가된 P28L/Y173F αTS은 야생형과 비슷한 구조로 회복되었다. 본 연구는 소수성 코아에 위치하는 Tyr¹⁷³ 잔기처럼 응집을 유도하는 여러 다른 잔기 자리를 보편적으로 억제하는 잔기가 존재할 수 있음을 시사해 준다.

• Bulletin of the Korean Chemical Society
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• 제31권4호
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• pp.1029-1030
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• 2010