• Journal of Veterinary Clinics
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• v.12 no.1
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• pp.905-910
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• 1995

GC 함유량(72%)이 높은 Mycobacterium paratuberculosis의 DNA의 PCR 증폭시 특이성과 생산성을 높이기 위하여 PCR 반응액에 denaturant인 DMSO, glycerol, formamide, Tween 20과 NP 40를 첨가하였다. Denaturant를 첨가하지 않은 상태의 PCR에서는 다수의 비특이적인 DNA가 관찰되었으며 표적 DNA 생산량이 낮았다. 모든 denaturant는 PCR의 특이성과 생산물의 생산량을 증가시했으며, 이들 중 DMSO, glycerol, farmamide와 NP 40는 높은 농도에서 생산량을 증가시켰다. Tween 20은 낮은 농도에서 생산량을 증가시켰다. Denaturant를 첨가하였음에도 불구하고 대부분의 반응에서 1 또는 2개의 비특이적인 DNA가 관찰되었다.

• Journal of the Korean Chemical Society
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• v.40 no.6
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• pp.394-400
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• 1996

The conformational transitions of oligopeptide -(HPPHPPP)$_n$- (H: hydrophobic amino acid, P: polar amino acid) which becomes cross linked-dimer are detected at various temperatures, pHs, ionic strengths, and the concentrations of denaturant. In this study, the transitions of oligopeptide due to denaturant, and those due to temperature are theoretically studied. Oligopeptide 20R as chain-dimer and guanidum-HCl as denaturant are used(20R, which contains 10 interchain and /or 10 intrachain electrostatic repulsions). Alpha helix-coil transitions by denaturant are very steep. This shows that the denaturations have transition states of, presumably all helical forms and random coils. The transitions by temperature are smoother than those by the concentration of denaturant. At low temperature the oligopetides which contain long helices exist more than those which contain short helices. As temperature rises, the mole fractions of the partially denatured oligopeptides increase. So the partially denatured oligopeptides are widely distributed at the transition temperature.

• Journal of Microbiology and Biotechnology
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• v.14 no.1
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• pp.153-157
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• 2004

Tyrosine phenol-lyase (TPL) is a useful enzyme for the synthesis of pharmaceutical aromatic amino acids. In the current study, sequential DNA shuffling and screening were used to enhance the stability of TPL. Twenty-thousand mutants were screened, and several improved variants were isolated. One variant named A13V, in which the $13^{th}$ amino acid alanine was substituted by valine, exhibited a higher temperature and denaturant stability than the wild-type TPL. The purified mutant TPL, A13V, retained about 60% of its activity at $76^\circ{C}$, whereas the activity of the wild-type TPL decreased to less than 20% at the same temperature. Plus, A13V exhibited about 50% activity with 3 M urea, while the wild-type TPL lost almost all its catalytic activity, indicating an increased denaturant tolerance in the mutant A13V. It is speculated that the substitution of Val for the Ala in the $\beta$-strand of the N-terminal arm was responsible for the heightened stabilization, and that the current results will contribute to further research on the structural stability of TPL.

• Proceedings of the Korean Nuclear Society Conference
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• 2001.05a
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• pp.71-71
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• 2001

• Archives of Pharmacal Research
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• v.12 no.2
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• pp.143-147
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• 1989

The urea effect on skin permeation of clonidine was investigated to reduce a log time and to increase a permeability. ICR mouse skin and human skin were used and were assumed to be a two-layer membrane consisted of stratum corneum and viable epidermis. The urea acted as a skin denaturant and humectant in the whole epidermis. Also it enhanced the skin permeability of clonidine about 3.5 times. On the other hand, it enhanced the skin permeability by acting as a humectant in the viable epidermis. But the urea effect on the whole epidermis was shown to be greater than that on the viable epidermis. Therefore, it was found that the effect of urea was greater on the stratum corneum than the viable epidermis. Variation of enhancing effect according to the concentration of urea was not found in the range of 1% to 20%.

• BMB Reports
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• v.39 no.1
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• pp.55-60
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• 2006

We describe a phage display strategy, based on the differential resistance of proteins to denaturant-induced unfolding, that can be used to select protein variants with improved conformational stability. To test the efficiency of this strategy, wild-type and two stable variants of ${\alpha}_1$-antitrypsin (${\alpha}_1AT$) were fused to the gene III protein of M13 phage. These phages were incubated in unfolding solution containing denaturant (urea or guanidinium chloride), and then subjected to an unfavorable refolding procedure (dialysis at $37^{\circ}C$). Once the ${\alpha}_1AT$ moiety of the fusion protein had unfolded in the unfolding solution, in which the denaturant concentration was higher than the unfolding transition midpoint ($C_m$) of the ${\alpha}_1AT$ variant, around 20% of the phage retained binding affinity to anti-${\alpha}_1AT$ antibody due to a low refolding efficiency. Moreover, this affinity reduced to less than 5% when 10 mg/mL skimmed milk (a misfolding-promoting additive) was included during the unfolding/refolding procedure. In contrast, most binding affinity (>95%) remained if the ${\alpha}_1AT$ variant was stable enough to resist unfolding. Because this selection procedure does not affect the infectivity of M13, the method is expected to be generally applicable to the high-throughput screening of stable protein variants, when activity-based screening is not possible.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.2
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• pp.122-127
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• 2005

A size exclusion chromatography (SEC) process, in the presence of denaturant in the refolding buffer was developed to refold recombinant human $interferon-\gamma$ ($rhIFN-\gamma$) at a high concentration. The $rhlFN-\gamma$ was overexpressed in E. coli resulting in the formation of inactive inclusion bodies (IBs). The IBs were first solubilized in 8 M urea as the denaturant, and then the refolding process performed by decreasing the urea concentration on the SEC column to suppress protein aggregation. The effects of the urea concentration, protein loading mode and column height during the refolding step were investigated. The combination of the buffer-exchange effect of SEC and a moderate urea concentration in the refolding buffer resulted in an efficient route for producing correctly folded $rhIFN-\gamma$, with protein recovery of $67.1\%$ and specific activity up to $1.2\times10^7\;IU/mg$.

• Biotechnology and Bioprocess Engineering:BBE
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• v.6 no.6
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• pp.410-418
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• 2001

The effects of several variables on the refolding of hen egg white lysozyme have been studied, Lysozyme was denatured in both urea, and guanidine hydrochloride(GuHCl), and batch refolded by dilution (100 to 1000 fold) into 0.1 M Tris-HCI, pH 8.2 mM EDTA 3 mM reduced glutathione and 0.3 mM oxidised glutathions. Refolding was found to be sensitive to temperature, with the highest refolding yield obtained at 50$\^{C}$. The apparent activation energy for lysozyme re-folding wasf ound to be 56kJ/mol, Refolding by dilution results in low concentrations of both de-naturant and reducing agent species. It was found that the residual concentrations obtained dur-ing dilution(100-fold dilution:[GuHCI]=0.06 mM, [DTT]=0.15 mM) were significant and could inhibit lysozyme refolding. This study has also shown that the initial protein concentration (1-10mg/mL) that is refolded is an important parameter. In the presence of residual GuHCl and DTT higher refolding yields were obtained when starting from higher initial lysozyme concentra-tions. This trend was reversed when residual denaturant components were removed from the re-folding buffer.

• Journal of Life Science
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• v.24 no.5
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• pp.558-566
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• 2014

Growth induced by cadmium (Cd) and ethylsalicylic acid (ESA) and the effect of ESA on rubisco/rubisco activase were studied in tobacco. The effect of denaturants on rubisco/rubisco activase was also investigated. In order to determine optimal concentration of ESA for growth of tobacco, tobacco was treated with $10^{-6}$-10 mM. It was found that its growth was the highest at $10^{-4}$ mM ESA. In the experiment using control, Cd treated group, ESA treated group, and Cd and ESA mixture group, ESA alone showed the highest growth and Cd showed the lowest growth. Cd treated group was the lowest in both rubisco/rubisco activase content and activity. ESA reduced the rubisco/rubisco activase content, but increased their activity. The activity of rubisco was inhibited by treating L-cysteine, urea, thiourea, ${\beta}$-mercaptoethanol, and EDTA other than guanidine-HCl in control group. L-cysteine, urea, thiourea, and guanidine-HCl treatments showed no change, but ${\beta}$-mercaptoethanol and EDTA increased rubisco activase activity. In conclusion, ESA inhibited the content of rubisco and promoted its activity, whereas promoted the content of rubisco activase and inhibited its activity. In addition, the content and activity of rubisco and rubisco activase inhibited by Cd were recovered by ESA. The activity of rubisco and rubisco activase by Cd and ESA was inhibited by the denaturant and the recovery of ESA inhibited by Cd was lost by the denaturant.

• Korean Chemical Engineering Research
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• v.43 no.2
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• pp.187-201
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• 2005

Bioprocessing technologies utilizing 'biorecognition' between a solid matrix and a protein is being widely experimented as a means to replacing the conventional, solution-based technology. Frequently the matrices are chromatographic resins with specific functional groups exposed outside. Since the reactions of and interactions with the proteins occur as they are attached to the solid matrix, this 'solid-phase' processing has distinct advantages over the solution-phase technology. Solid-phase refolding of inclusion body proteins uses ion exchange resins to adsorb denaturant-dissolved inclusion body. As the denaturant is slowly removed from the micromoiety around the protein, it is refolded into a native, three-dimensional structure. Once the refolding is complete, the folded protein can be eluted by a conventional elution technique such as the salt-gradient. This concept was successfully extended to 'EBA (expanded bed adsorption)-mediated refolding,' in which the denaturant-dissolved inclusion body in whole cell homogenate is adsorbed to a Streamline resin while cell debris and other impurity proteins are removed by the EBA action. The adsorbed protein follows the same refolding steps. This solid-phase refolding process shows the potential to improve the refolding yield, reduce the number of processing steps and the processing volume and time, and thus improve the overall process economics significantly. In this paper, the experimental results of the solid-phase refolding technology applied to several biopharmaceutical proteins of various types are presented.