• Journal of Microbiology and Biotechnology
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• v.6 no.6
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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.

• Proceeding of EDISON Challenge
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• 2015.03a
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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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• v.36 no.2
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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.

• Journal of the Korean Magnetic Resonance Society
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• v.24 no.3
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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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• v.65 no.3
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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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• v.35 no.6
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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.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2001.06a
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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.

• Proceedings of the Korean Society of Postharvest Science and Technology of Agricultural Products Conference
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• 2003.10a
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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.

• Journal of Life Science
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• v.32 no.9
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• pp.729-733
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• 2022

Aggregation of normally soluble proteins can cause disease-related problems. Tryptophan synthase α-subunit (αTS) in E. coli adopts one of most popular structural scaffolds, the TIM barrel fold. Previous mutagenesis of the αTS gene resulted in many aggregation-prone mutant proteins. Here, Y173F (Tyr at residue 173 to Phe) substitution, which imparts increased stability, was tested for its ability to suppress aggregation of aggregation-prone mutant proteins (Y4C, S33L, P28L, P28S, G44S, D46N, P96L, and P96S). Aggregation was suppressed in all eight severe aggregate-forming mutants (all differing in their mutation positions), by the Y173F replacement. P28L αTS, which was available in pure form, was further analyzed and showed reduced secondary structure content, lower stability, and a looser structure with more exposed hydrophobic surface compared to the wild type protein. A double mutant P28L/Y173F protein showed almost no indication of these changes compared to the wild type protein. We hypothesized that Tyr at position 173 in αTS is positioned at the hydrophobic core and may serve to suppress the aggregation of this protein caused by other residues. Important residue (s) could be working widely in the prevention/suppression of protein aggregation.

• Bulletin of the Korean Chemical Society
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• v.31 no.4
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• pp.1029-1030
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• 2010