• Journal of the Korean Society for Aeronautical & Space Sciences
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• v.35 no.10
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• pp.905-911
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• 2007

In this paper, geometrically and materially non-linear finite element analyses were performed to study the crease behavior of thin membranes. The cross-section of the membrane was modeled with 2-dimensional plane strain elements. To simulate the creasing process, the membrane mesh was folded, compressed to prescribed crease gauge by activating two rigid contact surfaces, and then released to give the crease topology. Various crease gauges were considered to investigate the effect of crease intensity on the initial deployment angle. The crease geometry was also obtained by experiments and the results were compared.

• 한국생물공학회:학술대회논문집
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• 2002.04a
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• pp.485-488
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• 2002

재접힘 고체상 재접힘은 높은 재현성을 보였으며 고체상 재접힘된 단백질은 Native와 같은 구조를 형성하였다. 따라서 이 연구는 고체상 재접힘 방법이 분자간의 상호작용을 억제하는 것이 응집현상을 탈피하게된 결과 일 것이라는 것에 의해 재접힘 수율을 높일 수 있다고 기대한다.

• KSBB Journal
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• v.17 no.2
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• pp.153-157
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• 2002

A fusion protein, consisting of a human epidermal growth factor as the recognition domain and human angiogenin as the toxin domain, can be used as a targeted therapeutic against breast cancer cells among others. The fusion protein was expressed as an inclusion body in recombinant E. coli, yet when the conventional solution-phase refolding process was used the refolding yield was very low due to severe aggregation, probably because of the opposite surface charge resulting from the vastly different pl values of each domain. Accordingly the solid-phase refolding process, which exploits the ionic interactions between a solid matrix and the protein, was tried, however the ionic binding yield was also very low regardless of the resins and pH conditions used. Therefore, to provide a higher affinity toward the solid matrix, six Iysine residues were tagged to the N-terminus of the hEGF domain. When cation exchange resins, such as heparin- or CM-Sepharose, were used as the matrix, the adsorption capacity increased 2.5~3-fold and the subsequent refolding yield increased nearly 15-fold compared to the conventional process. A similat result was also obtained when an Ni-NTA metal affinity resin was used.

• KSBB Journal
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• v.18 no.6
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• pp.500-505
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• 2003

‘LK (lipoprotein kringle) 68’is a polypeptide of a modified ansiostatin consisting of three kringle structures that might be clinically useful as a potential cancer therapeutics. It can be produced by overexpressing it as inclusion body in recombinant E. coli. In this study, solid-phase refolding processes using packed bed adsorption (PBA) and expanded bed adsorption (EBA) column were carried out to compare their refolding yields with that of the conventional, solution-phase refolding process, For the solution-phase and the PBA-mediated processes employing Q-Sepharose, washed inclusion body was used as the starting material, whereas both washed inclusion body and E. coli homogenate were used for the EBA-mediated process employing streamline DEAE. On the final recovery LK68 per unit mass of wet cell basis, the EBA- and PBA-mediated processes showed about 2.7- and 1.5-fold higher yields, respectively, than the solution-phase refolding method. The solid-phase refolded LK68 demonstrated the same Iysine binding bioactivity and the retention time in the RP-and SEC-HPLC as those of the native protein.

• 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.

• 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.

• KSBB Journal
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• v.20 no.5 s.94
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• pp.338-340
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• 2005

Covalent modification of a protein with polyethylene glycol (PEG) has become one of the most widely used and well established drug enhancement strategies in the biopharmaceutical industry. The general benefits enjoyed by PEGylation, such as prolonged serum half-lives or reduced immunogenicity in vivo, are well known. By now the PEGylation process has been performed with purified proteins, and it is required to recover the desired PEGylate by a multi-step purification process. The ultimate aim of our research is to develop an integrated process of PEGylation and in vitro refolding starting with inclusion body material. For this, we investigated the feasibility that a protein could be PEGylated under a denaturing condition and also the PEGylated proteins could be refolded correctly. Using lipase as a model protein, we found that it was PEGylated in the presence of 8M urea and that the PEG molecules covalently attached to lipase did not appear to hinder its refolding.

• 한국생물공학회:학술대회논문집
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• 2003.04a
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• pp.480-483
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• 2003

Fusion ferritin$(F_H+F_L)$, an iron-binding protein, was purified from recombinant E. coli by two-step sonications with urea. Unfolded ferritin was refolded by gel filtration chromatography with various concentration of urea. 50 mM Tris-HCl(pH 8.0) buffers with 1 M to 4 M urea were used in GFC. Objective was to characterize the structure change with urea concentration. Molecular weight was determined using GF-HPLC and RP-HPLC was used to quantify the unfolded and refolded proteins.

• 한국생물공학회:학술대회논문집
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• 2003.04a
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• pp.513-516
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• 2003

고체상 풀림과 재접힘 공정을 통해서 EK의 활성이 회복되어지는 것을 확인할 수 있었다. 이는 고정화 효소를 사용함으로써 기존의 액상반응에서 불가능한 효소의 재사용 문제를 해결할 수 있다. 친화도보다는 다중 공유결합을 통한 효소의 고정화가 효소의 활성 회복에 높은 안정성을 가질 수 있다고 예상한다.

• Proceedings of the Korea Technical Association of the Pulp and Paper Industry Conference
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• 2008.04a
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• pp.282-289
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• 2008