• Journal of the Korean Chemical Society
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• v.49 no.5
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• pp.479-487
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• 2005

• KSBB Journal
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• v.14 no.6
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• pp.651-654
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• 1999

Using recombinant human growth hormone as a model protein, we carried out unfolding by adding a denaturant such as urea, guanidine HCl, or SDS followed by refolding by dilution and dialysis. The objectives were to monitor the structural changes during in vitro refolding process and, based on the results, to develop a quantitative method of refolding progress assessment. The changes in surface hydrophobicity were measured by fluorescence tagging of 1-anilinonaphthalene-8-sulfonate(1,8-ANS) to the hydrophobic portions, and those in the secondary structure were monitored by using far UV-CD(circular dichroism) spectroscopy. Also, we used RP-HPLC to separate and quantify the folded and unfolded proteins to correlate the result with the structure analysis. Our results indicate the surface hydrophobicity are well correlated with the formations of the secondary structure, primarily ${\alpha}$-helices, as well as the disulfide bridges. We expect this monitoring technique can be applied in industrial fields as a means to quantitatively assess the progress of in-vitro refolding of recombinant proteins.

• Proceedings of the KAIS Fall Conference
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• 2003.06a
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• pp.328-330
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• 2003

A surface equation of state for globular proteins at air-water interface accounting for the molecular structure, segment-segment, segment-solvent, and electrostatic interactions was proposed and compared to C-14 isotope experiments. This lattice model comprised a simplifying assumption that all adsorbed segments are in the form of trains. The number of segment adsorbed per molecule in case of bovine serum albumin linearly depended on the surface concentration whereas the lysozyme segments adsorbed at the interface were independent of surface concentration. The segment-solvent(water) interaction for both of proteins were found to be unfavorable owing to the proteins unfolding. From comparison of model computation and experimental data, BSA unfolded more than lysozyne because of the larger surface area of contact.

• KSBB Journal
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• v.15 no.1
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• pp.42-48
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• 2000

Urokinase (UK), a thrombolytic enzyme used to clear catheters obstructed by blood clots, can be also used industrially in the recombinant protein purification system to cleave a fusion protein linked with a certain fragment of GST. We have immobilized UK by covalent attachment to activated Sepharose 6B-Cl gels and evaluated its performance to cleave a fusion protein of hGH and GST. The Sepharose gels were activated by etherification with glycidol (2,3-epoxypropanol) and further oxidized with periodate resulting in glyceryl-Sepharose gels. After the activation treatment, surface density of the aldehyde groups was 7-30 $\mu$mol-aldehde/mL-gel. Immobilization yield was higher than 99% at high pH (10.5), and the immobilized UK maintained ca. 80% specific activity of the soluble UK. In a column reaction the cleavage yield heavily depended on the feed rate, and it was nearly 86% of that from soluble UK. And the immobilized UK was successfully regenerated by unfolding and refolding with 6M GuHCl. After cleavaging reaction, the monomeric hGH was purified by using expanded bed adsorption chromatography.

• Journal of the Korean Chemical Society
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• v.58 no.6
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• pp.560-568
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• 2014

The contribution of electrostatic interactions to protein folding reaction was studied by using mutant ubiquitin with lysine to alanine mutation at residue position 29. Based on the three dimensional structure of ubiquitin, lysine 29 is located close to negatively charged glutamate 16 and aspartate 21 and considered to stabilize the native state of ubiquitin by electrostatic interactions between these residues. The equilibrium unfolding experiment showed that the native stability was decreased by about ~20% upon mutation. This observation indicates lysine 29 indeed forms electrostatic interactions with nearby residues. Folding kinetics measurements using stopped-flow device and quantitative analysis of kinetics data indicate that ubiquitin folds from unfolded state to native state via intermediate state as observed previously. This intermediate state was observed to form immediately after the initiation of folding reaction. The folding intermediate was shown to be destabilized considerably upon lysine to alanine mutation. These observations indicate that electrostatic interactions can form early stage of protein folding and hence lead the folding reaction.