• Journal of Life Science
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• v.23 no.2
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• pp.319-323
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• 2013

Protein aggregation can cause diseases and hinder the production of useful recombinant proteins. The present study showed that at least three types of aggregates can be formed from tryptophan synthase ${\alpha}$-subunit (${\alpha}TS$) by varying conditions: (1) an opaque white precipitous aggregate, (2) a transparent gel-like precipitous aggregate, and (3) an unprecipitous aggregate. Macroscopically different aggregate types might suggest different mechanisms underlying aggregation processes.

• YAKHAK HOEJI
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• v.45 no.6
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• pp.656-663
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• 2001

The quality of an intravenous immunoglobulin preparation (IVIG) is reflected by the degree of nonspecific activation of complements, the so-called anticomplementary activity (ACA). ACA of aggregates in IVIG was investigated using method by the European Pharmacopoeia and Clq-coated microtiter enzyme-linked immunosorbent assay (ELISA). Both the EP method and the ELISA method showed a dose response curve with the amount of complements bound increasing with the percentage content of aggregates in immunoglobulin standard. The correlation between the two tests was good (r=0.96, r=0.99). However, the correlation was not found when the ACA (EP method) of IVIG product was compared with its aggregate percentage. These results emphasize that the method of aggregate formation affects ACA and that estimation of the percentage distribution of aggregates by HPLC may not reflect ACA. In analysing WIG product for Clq binding activity test with the ELISA, the result by using Protein A-HRP correlated with aggregate percentage (r=0.84). But the correlation decreased (r=0.48) when the result used Protein A-AP(having poorer sensitivity than HRP) was compared with aggregate percentage. As a result, some variation between the two methods, due to differences in assay principles, is to be expected. However, ELISA technique has the advantage in that it is easier to perform, more precise and less subject to reagent variability, and is the more suitable screening method than HPLC analysis.

• Journal of Life Science
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• v.9 no.3
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• pp.328-332
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• 1999

The effect of cations on the formation of protein aggregates was examined by in vitro refolding of mutant tryptophan synthase $\alpha$-subunit in which Pro 24 was replaced by Leu. $NH^{4+},; K{^+}; and; Na^{+}$ and no effect, but $Mg^{2+}; and; Ca^{2+}$stimulated the formation of protein aggregates in dose-dependent manner. It is suggested that $Mg^{2+} and Ca^{2+}$ may be implicated in the formation of protein aggregates in vivo.

• Journal of Life Science
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• v.11 no.1
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• pp.43-47
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• 2001

A mutant tryptophan synthase $\alpha$-subunit, where Pro28 was replaced with Leu, tends to be expressed in recombinant E. coli. CD and fluorescence spectra of this protein indicate some changes in secondary and tertiary structure. Wild type protein was more or less affected by {TEX}$Ca^{2+}${/TEX} ion in regards of the fluorescent properties of its native, unfolded and intermediate forms, but the mutant protein was not at all. The dramatic structural changes may be related to the aggregation of this mutant protein.

• Restorative Dentistry and Endodontics
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• v.39 no.3
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• pp.187-194
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• 2014

Objectives: The effects of bone morphogenetic protein-2 (BMP-2) and enamel matrix derivative (EMD) respectively with mineral trioxide aggregate (MTA) on hard tissue regeneration have been investigated in previous studies. This study aimed to compare the osteogenic effects of MTA/BMP-2 and MTA/EMD treatment in MC3T3-E1 cells. Materials and Methods: MC3T3-E1 cells were treated with MTA (ProRoot, Dentsply), BMP-2 (R&D Systems), EMD (Emdogain, Straumann) separately and MTA/BMP-2 or MTA/EMD combination. Mineralization was evaluated by staining the calcium deposits with alkaline phosphatase (ALP, Sigma-Aldrich) and Alizarin red (Sigma-Aldrich). The effects on the osteoblast differentiation were evaluated by the expressions of osteogenic markers, including ALP, bone sialoprotein (BSP), osteocalcin (OCN), osteopontin (OPN) and osteonectin (OSN), as determined by reverse-transcription polymerase chain reaction analysis (RT-PCR, AccuPower PCR, Bioneer). Results: Mineralization increased in the BMP-2 and MTA/BMP-2 groups and increased to a lesser extent in the MTA/EMD group but appeared to decrease in the MTA-only group based on Alizarin red staining. ALP expression largely decreased in the EMD and MTA/EMD groups based on ALP staining. In the MTA/BMP-2 group, mRNA expression of OPN on day 3 and BSP and OCN on day 7 significantly increased. In the MTA/EMD group, OSN and OCN gene expression significantly increased on day 7, whereas ALP expression decreased on days 3 and 7 (p < 0.05). Conclusions: These results suggest the MTA/BMP-2 combination promoted more rapid differentiation in MC3T3-E1 cells than did MTA/EMD during the early mineralization period.

• 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.

• Biomedical Science Letters
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• v.9 no.4
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• pp.203-207
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• 2003

Protein aggregation could be problematic as causes of diseases and hindrance in the production of useful recombinant proteins. Aggregation of mutant tryptophan synthase $\alpha$-subunits was examined by treatment with urea and at high temperature. Large amorphous aggregate seemed to appear by heat treatment, while more various aggregates in size were formed by treatment with urea at low concentration. The result indicates that different aggregate in size could be formed depending on the treatment condition, suggesting different mechanisms underlying aggregation processes.

• Molecules and Cells
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• v.42 no.6
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• pp.480-494
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• 2019

Aggregates of disease-causing proteins dysregulate cellular functions, thereby causing neuronal cell loss in diverse neurodegenerative diseases. Although many in vitro or in vivo studies of protein aggregate inhibitors have been performed, a therapeutic strategy to control aggregate toxicity has not been earnestly pursued, partly due to the limitations of available aggregate models. In this study, we established a tetracycline (Tet)-inducible nuclear aggregate (${\beta}23$) expression model to screen potential lead compounds inhibiting ${\beta}23$-induced toxicity. High-throughput screening identified several natural compounds as nuclear ${\beta}23$ inhibitors, including peucedanocoumarin III (PCIII). Interestingly, PCIII accelerates disaggregation and proteasomal clearance of both nuclear and cytosolic ${\beta}23$ aggregates and protects SH-SY5Y cells from toxicity induced by ${\beta}23$ expression. Of translational relevance, PCIII disassembled fibrils and enhanced clearance of cytosolic and nuclear protein aggregates in cellular models of huntingtin and ${\alpha}$-synuclein aggregation. Moreover, cellular toxicity was diminished with PCIII treatment for polyglutamine (PolyQ)-huntingtin expression and ${\alpha}$-synuclein expression in conjunction with 6-hydroxydopamine (6-OHDA) treatment. Importantly, PCIII not only inhibited ${\alpha}$-synuclein aggregation but also disaggregated preformed ${\alpha}$-synuclein fibrils in vitro. Taken together, our results suggest that a Tet-Off ${\beta}23$ cell model could serve as a robust platform for screening effective lead compounds inhibiting nuclear or cytosolic protein aggregates. Brain-permeable PCIII or its derivatives could be beneficial for eliminating established protein aggregates.

• BMB Reports
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• v.33 no.3
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• pp.256-262
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• 2000

In this study the disassembly process of chlorophyII (ChI)protein complexes of a stay-green mutant (ore10 of Arabidopsis thaliana) was investigated during the dark incubation of detached leaves. During this dark-induced senescence (DIS), the Chi loss was delayed in the mutant, while the photochemical efficiency of photosystem II (PSII) or Fv/Fm was accelerated when compared with the wild type (WT) leaves. This indicates that the decrease in Fv/Fm is a separate process and not causally-linked to the degradation of Chi during DIS of Arabidopsis leaves. In the native green gel electrophoresis of the Chi-protein complexes, which was combined with an additional twodimensional SDS-PAGE analysis, the delayed senescence of this mutant was characterized by the appearance of an aggregate at 1 d or 2 d, as well as very stable light harvesting complex II (LHCII) trimers until 5 d after the start of DIS. The polypeptide composition of the aggregates varied during the whole DIS at 5 d. Dl protein appeared to be missing in the aggregates. This result supports the idea of a faster depletion of functional PSH in the mutants compared with WT, as suggested by the earlier reduction of Fv/Fm and the stable Chl a/b ratio in the mutants. At 5 d, the WT leaves also often showed aggregates, but the polypeptide composition was different from those of ore10. The results presented suggest that the formation of aggregates, or stable LHCII trimers in the stay-green mutants, is a way to structurally protect Chi-protein complexes from serious proteolytic degradation. Detailed disassembly processes of Chi-protein complexes in WT and ore10 mutants are discussed.

• Journal of Microbiology and Biotechnology
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• v.21 no.11
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• pp.1159-1165
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• 2011

Biodiesel is methyl and ethyl esters of long-chain fatty acids produced from vegetable oils or animal fats. Lipase enzymes have occasionally been used for the production of this biofuel. Recently, biodiesel production using immobilized lipase has received increased attention. Through enhanced stability and reusability, immobilized lipase can contribute to the reduction of the costs inherent to biodiesel production. In this study, methanol-tolerant lipase M37 from Photobacterium lipolyticum was immobilized using the cross-linked enzyme aggregate (CLEA) method. Lipase M37 has a high lysine content (9.7%) in its protein sequence. Most lysine residues are located evenly over the surface of the protein, except for the lid structure region, which makes the CLEA preparation yield quite high (~93%). CLEA M37 evidences an optimal temperature of $30^{\circ}C$, and an optimal pH of 9-10. It was stable up to $50^{\circ}C$ and in a pH range of 4.0-11.0. Both soluble M37 and CLEA M37 were stable in the presence of high concentrations of methanol, ethanol, 1-propanol, and n-butanol. That is, their activities were maintained at solvent concentrations above 10% (v/v). CLEA M37 could produce biodiesel from olive oil and alcohols such as methanol and ethanol. Additionally, CLEA M37 generated biodiesel via both 2-step methanol feeding procedures. Considering its physical stability and reusability, CLEA M37 may potentially be used as a catalyst in organic synthesis, including the biodiesel production reaction.