• Bulletin of the Korean Chemical Society
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• 제31권10호
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• pp.2877-2883
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• 2010

The folding kinetics of $WT^*$ ubiquitin variant with valine to alanine mutation at sequence position 26 (HubWA) was studied by stopped-flow fluorescence spectroscopy. While unfolding kinetics showed a single exponential phase, refolding reaction showed three exponential phases. The semi-logarithmic plot of urea concentration vs. rate constant for the first phase showed v-shape pattern while the second phase showed v-shape with roll-over effect at low urea concentration. The rate constant and the amplitude of the third phase were constant throughout the urea concentrations, suggesting that this phase represents parallel process due to the configurational isomerization. Interestingly, the first and second phases appeared to be coupled since the amplitude of the second phase increased at the expense of the amplitude of the first phase in increasing urea concentrations. This observation together with the roll-over effect in the second folding phase indicates the presence of intermediate state during the folding reaction of HubWA. Quantitative analysis of Hub-WA folding kinetics indicated that this intermediate state is on the folding pathway. Folding kinetics measurement of a mutant HubWA with hydrophobic core residue mutation, Val to Ala at residue position 17, suggested that the intermediate state has significant amount of native interactions, supporting the interpretation that the intermediate is on the folding pathway. It is considered that HubWA is a useful model protein to study the contribution of residues to protein folding process using folding kinetics measurements in conjunction with protein engineering.

• 농업과학연구
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• 제41권3호
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• pp.237-243
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• 2014

Human Glucagon like peptide-1 (GLP-1) is an incretin hormone that promotes secretion of insulin. In order to eliminate the formation of the soluble aggregate, Ala19 in GLP-1 was substituted with Thr, resulting in a GLP-1 mutant GLP-1A19T. The gene synthesis of GLP-1A19T and the fusion of 6-lysine tagged ubiquitin gene were accomplished by using the overlap extension polymerase chain reaction. The ubiquitin fused GLP-1A19T (K6UbGLP-1A19T) is expressed as form of inclusion body with little formation of the soluble aggregation in recombinant E. coli. In order to produce K6UbGLP-1A19T in large amounts, fed-batch fermentation was carried out in a pH-stat feeding strategy. Maximum dry cell weight of 87.7 g/L and 20.4% of specific K6UbGLP-1A19T content were obtained. Solid-phase refolding using a cation exchanger was carried out to renature K6UbGLP-1A19T. The refolded K6UbGLP-1A19T aggregated little and was released GLP-1A19T by on-column cleavage with ubiquitin-specific protease-1. The molecular mass of GLP-1A19T showed an accurate agreement with its theoretical molecular mass.

• Journal of Plant Biotechnology
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• 제49권1호
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• pp.15-29
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• 2022

Cyclophilins (CYPs) are highly conserved ubiquitous proteins belong to the peptidyl prolyl cis/trans isomerase (PPIase) superfamily. These proteins are present in a wide range of organisms; they contain a highly conserved peptidyl-prolyl cis/trans isomerase domain. A comprehensive database survey identified a total of 35 genes localized in all cellular compartments of Solanum lycopersicum L., but largely in the cytosol. Sequence alignment and conserved motif analyses of the SlCYP proteins revealed a highly conserved CLD motif. Evolutionary analysis predicted the clustering of a large number of gene pairs with high sequence similarity. Expression analysis using the RNA-Seq data showed that the majority of the SlCYP genes were highly expressed in mature leaves and blooming flowers, compared with their expression in other organs. This study provides a basis for the functional characterization of individual CYP genes in the future to elucidate their role(s) in protein refolding and long-distance signaling in tomatoes and in plant biology, in general.

• BMB Reports
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• 제40권5호
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• pp.783-790
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• 2007

Receptor binding plays an important role in determining host specificity of the Bacillus thuringiensis Cry $\delta$-endotoxins. Mutations in domains II and III have suggested the participation of certain residues in receptor recognition and insect specificity. In the present study, we expressed the cloned domain II-III fragment of Cry4Ba and examined its binding characteristics to mosquito-larval midgut proteins. The 43-kDa Cry4Ba-domain II-III protein over-expressed in Escherichia coli as inclusion bodies was only soluble when carbonate buffer, pH 10.0 was supplemented with 4M urea. After renaturation via stepwise dialysis and subsequent purification, the refolded domain II-III protein, which specifically reacts with anti Cry4Ba-domain III monoclonal antibody, predominantly exists as a $\beta$-sheet structure determined by circular dichroism spectroscopy. In vitro binding analysis to both histological midgut tissue sections and brush border membrane proteins prepared from susceptible Aedes aegypti mosquito-larvae revealed that the isolated Cry4Ba-domain II-III protein showed binding functionality comparable to the 65-kDa full-length active toxin. Altogether, the data present the 43-kDa Cry4Ba fragment comprising domains II and III that was produced in isolation was able to retain its receptor-binding characteristics to the target larval midgut proteins.

• 생명과학회지
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• 제19권5호
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• pp.561-565
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• 2009

인간조직인자는 혈액응고인자 factor VII 과 복합체를 형성하며 연속적인 혈액응고 연쇄반응을 촉매하는 효소 활성체이다. 복합체 형성에 필수적인 이 조직인자의 세포 외 부분이, 기존의 융합 단백질 및 히스티딘 말단이 없는 새로운 발현 벡터에 의해 대장균 내에서 과량 발현 되었다. 봉입체 형태로 발현된 재조합 인간조직인자는 DEAE-Sephacel 크로마토그라피 기술을 적용하여 분리, 정제 및 구조적 복원이 동시에 시도 되었다. 정제된 재조합 단백질은 SDS-PAGE 분석에서 순수한 형태로 나타났으며, 생물학적 활성도 또한 기존의 조직인자와 거의 동등함을 보였다. 본 연구의 발현 및 정제 시스템은 이전의 보고에서 보여진 방법들에 비해 단백질 분해효소를 사용하지 않아 추가적인 크로마토그라피 과정이 필요 없어 좀 더 효율적이기 때문에 기존의 발현 시스템에 대해 대체할 수 있는 매우 유용한 방법으로 제공된다.

• BMB Reports
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• 제33권5호
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• pp.407-411
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• 2000

The effects of heat shock, or all-trans retinoic acid, on the expression of the C-reactive protein mRNA in the HT-29 human colon carcinoma cells, as well as the functional role of the C-reactive protein as a molecular chaperone, were studied. The expression level of the C-reactive protein mRNA in the HT-29 cells was increased time-dependently when exposed to heat-shock, and dose-dependently when treated with all-trans retinoic acid. The activities of transglutaminase C and K in the HT-29 cells were significantly increased when treated with all-trans retinoic acid. The C-reactive protein prevented thermal aggregation of the citrate synthase and stabilized the target enzyme, citrate synthase. The C-reactive protein promoted functional refolding of the urea-denatured citrate synthase up to 40-70%. These results suggest that the C-reactive protein, which is induced in human colon carcinoma cells, when heated or treated with all-trans retinoic acid has in a part functional activity of the molecular chaperone.

• Bulletin of the Korean Chemical Society
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• 제32권6호
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• pp.1925-1930
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• 2011

Psychrophilic bacteria have acquired cold-resistance in order to protect themselves against freezing temperatures, which would otherwise be lethal. DnaK/DnaJ/GrpE systems are molecular chaperones which facilitate proper folding of newly synthesized proteins. Efficient folding processes are of great importance especially in a cold environment, such as the Arctic. In order to understand the protection mechanisms of psychrophilic bacteria against cold temperatures, we have explored a genome of KOPRI22215, tentatively identified as Psychromonas arctica, whose genome sequence has not yet been discovered. With an aim of searching for a coding gene of DnaK from KOPRI22215, we have applied a series of polymerase chain reactions (PCR) with homologous primers designed from other Psychromonas species and LA PCR in vitro cloning. 1917 bp complete coding sequence of dnaK from KOPRI22215 was identified including upstream promoter sites. Recombinant plasmids to overexpress PaDnaK along with EcDnaK (DnaK of E. coli) were then constructed in pAED4 vector and the pET-based system to induce PaDnaK expression by IPTG. Characterization assays of expressed PaDnaK were carried out by measuring survival rates upon 4 day incubation at 4 $^{\circ}C$: a refolding assay as molecular chaperone, and ATPase assay for functional activity. Taking account of all the data together, we conclude that PaDnaK was identified, successfully expressed, and found to be more efficient in providing cold-resistance for bacterial cells.

• BMB Reports
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• 제33권2호
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• pp.120-125
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• 2000

M2PI is an active single chain mini-proinsulin with a 9-residue linker containing the turn-forming sequence 'YPGDV' between the B- and A-chains, but which retains about 50% of native insulin receptor binding activity. The refolding efficiency of M2PI is higher than proinsulin by 20-40% at alkaline pH, and native insulin is generated by the enzymatic conversion of M2PI. The solution structure of M2PI was determined by NMR spectroscopy. The global structure of M2PI is similar to that of native insulin, but the flexible linker between the B- and A-chains perturbed the N-terminal A-chain and C-terminal B-chain. The helix in the N-terminal A-chain is partly perturbed and the ${\beta}$-turn in the B-chain is disrupted in M2PI. However, the linker between the two chains was completely disordered indicating that the designed turn was not formed under the experimental conditions (20% acetic acid). Considering the fact that an insulin analogue, directly cross-linked between the C-terminus of the B-chain and the N-terminus of the A-chain, has negligible binding activity, a flexible linker between the two chains is sufficient to keep binding activity of M2PI, but the perturbed secondary structures are detrimental to receptor binding.

• BMB Reports
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• 제48권2호
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• pp.91-96
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• 2015

Cells express several antioxidant enzymes to scavenge reactive oxygen species (ROS) responsible for oxidative damages and various human diseases. Therefore, antioxidant enzymes are considered biomedicine candidates. Among them, extracellular superoxide dismutase (SOD3) had showed prominent efficacy against asthma and inflammation. Despite its advantages as a biomedicine, the difficulty in obtaining large quantity of active recombinant human SOD3 (rhSOD3) has limited its clinical applications. We found that a significant fraction of over-expressed rhSOD3 was composed of the inactive apo-enzyme and its potency against inflammation depended on the rate of metal incorporation. Also, purified rhSOD3 was unstable and lost its activity very quickly. Here, we suggest an ideal preparative method to express, purify, and store highly active rhSOD3. The enzymatic activity of rhSOD3 was maximized by incorporating metal ions into rhSOD3 after purification. Also, albumin or polyethylene glycol prevented rapid inactivation or degradation of rhSOD3 during preparative procedures and long-term storage.

• Journal of Microbiology and Biotechnology
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• 제19권12호
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• pp.1628-1634
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• 2009

Small heat shock proteins (sHSPs) function as molecular chaperones that protect cells against environmental stresses. In the present study, the genes of hsp17.6 and hsp17.7, cytosolic class I sHSPs, were cloned from a tropical plant, Ageratina adenophorum. Their C-terminal domains were highly conserved with those of sHSPs from other plants, indicating the importance of the C-terminal domains for the structure and activity of sHSPs. The recombinant HSP17.6 and HSP17.7 were applied to determine their chaperone function. In vitro, HSP17.6 and HSP17.7 actively participated in the refolding of the model substrate citrate synthase (CS) and effectively prevented the thermal aggregation of CS at $45^{\circ}C$ and the irreversible inactivation of CS at $38^{\circ}C$ at stoichiometric levels. The prior presence of HSP17.7 was assumed to suppress the thermal aggregation of the model substrate CS. Therefore, this report confirms the chaperone activity of HSP17.6 and HSP17.7 and their potential as a protectant for active proteins.