• Journal of Life Science
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• v.17 no.2 s.82
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• pp.211-217
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• 2007

The goal of this study is to investigate effects of temperature and co-chaperonin requirement for in vitro protein refolding assisted by E. coli chaperone GroEL under permissive and nonpermissive temperature conditions. In vitro protein refolding of two denatured proteins was kinetically investigated under several conditions in the presence of GroEL. Effects of temperature and GroES-requirement on the process of prevention of protein aggregation and refolding of denatured protein were extensively monitored. We have found that E. coli GroEL chaperone system along with ATP is required for invitro refolding of unfolded polypeptide under nonpermissive temperature of $37^{\circ}C$. However, under permissive condition spontaneous refolding can occur due to lower temperature, which can competes with chaperone-mediated protein refolding via GroEL chaperone system. Thus, GroEL seemed to divert spontaneous refolding pathway of unfolded polypeptide toward chaperone-assisted refolding pathway, which is more efficient protein refolding pathway.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.27 no.4
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• pp.335-343
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• 1994

To determine the optimal level of cryoprotectant on the denaturation of pollock surimi produced in Korea, the relative cryoprotective effects of crystalline sorbitol alone and in combination with sucrose were assessed. Freeze induced protein denaturation was also studied as affected by polyphosphates and maltodextrin during frozen storage at $-25^{\circ}C$ for 16 weeks. Variables evaluated included salt extractable protein, drip loss and in vitro protein quality. The best cryoprotective effect was achieved from sucrose/sorbitol 1:1(w/w) mixture at $8\%$ with $0.2\%$ sodiumpyrophosphate and sodiumtriphosphate(1:1, w/w) in surimi by measurement of salt extractable protein and drip loss. Those cryoprotectants had little effect on surimi protein quality during frozen storage as measured by trypsin inhibitor(TI), protein digestibility and computed protein efficiency ratio(C-PER). Protein digestibility of surimi was not changed significantly by polyphosphate and maltodextrin at various levels(p<0.05), with the exception of 4 or $6\%$ sorbitol and $10\%$ sucrose alone which resulted in a higher digestibility. $8\%$ sorbitol/sucrose (5:3, w/w) treatment without polyphosphates showed the highest cryoprotective effectiveness from digestibility assay.

• 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.

• Korean Journal of Food Science and Technology
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• v.16 no.3
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• pp.279-284
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• 1984

Microstructure of milk powder and cheese made from milk powder were observed by electron microscope. Freeze dried milk powder showed apple-like appearance. The cheese made from freeze dried milk powder had relatively flat surface and homogenous deposit in compare with classical processed cheese. Imported milk powder also indicated similar surface as well as freeze dried milk powder, however, the cheese made from imported milk powder had somewhat coarse surface structures with the spaces between casein matrix and deposit. Commericial milk powder showed irregular shape in size and coagulum which were possibly denatured in the course of drying. The cheese made from commercial powder indicted irregular and small deposit and porous structure. The porousity of the cheese seemed to be influenced by the degree of heat treatment. Denatured protein would be less dispersive than native in presence of polyphosphates. Fat globule and protein micelle of cheese made from skim milk powder get very adjacent to each other and showed compactness of micelles. It is thought that melting mechanism of skim milk powder was different from the melting of typical processed cheese.

• Proceedings of the Korean Society of Fisheries Technology Conference
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• 2002.10a
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• pp.73-74
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• 2002

수세공정을 거치지 않고 어육 단백질의 용해도 특성을 이용하여 pH 2.5 부근과 pH 10.5 부근에서 어육 단백질을 가용화 시킨 후 최소 용해도를 보이는 pH 5.2 부근에서 침전시켜 회수하고, pH를 7.0으로 조절한 후 법동 변성 방지제를 첨가하여 수리미를 조제하는 공정이 개발(Choi and Park, 2000; Yongsawatdigul and Park, 2001)됨에 따라 소실되는 근형질 단백질을 회수함으로서 수리미 수율을 최대로 증가시킬 수 있게 되었다(Kim et al., 2001, 2002; Choi et al., 2002). (중략)

• Proceedings of the Korean Society of Fisheries Technology Conference
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• 2002.10a
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• pp.71-72
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• 2002

수세공정을 거치지 않고 어육 단백질의 용해도 특성을 이용하여 pH 2.5 부근과 pH 10.5 부근에서 어육 단백질을 가용화 시킨 후 최소 용해도를 보이는 pH 5.2 부근에서 침전시켜 회수하고, pH를 7.0으로 조절한 후 법동 변성 방지제를 첨가하여 수리미를 조제하는 공정이 개발(Choi and Park, 2000; Yongsawatdigul and Park, 2001)됨에 따라 소실되는 근형질 단백질을 회수함으로서 수리미 수율을 최대로 증가시킬 수 있게 되었다(Kim et al., 2001, 2002: 최 등, 2002). (중략)

• Journal of the Korean Society of Food Science and Nutrition
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• v.33 no.10
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• pp.1668-1675
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• 2004

The effect of pH on surface hydrophobicity, sulfhydryl group, infrared spectrum, SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) pattern and enthalpy was investigated in recovered protein from mackerel and frozen blackspotted croaker by alkaline processing. Hydrophobic residue in myofibrillar protein exposed to the surface of protein, and hydrophobic interaction were the highest around 6$0^{\circ}C$. The surface hydrophobicity was different between myofibrillar protein and myofibrillar protein including sarcoplasmic protein (recovered protein). The peak at 1636 c $m^{-l}$ was increased with pH, and the recovered protein was unfolded in alkali pH. Difference of surface and total sulfhydryl group at pH 7.0 and 10 was comparative high, and decrease of surface sulfhydryl group indicated formation of S-S bonds. Mackerel and frozen blackspotted croaker in alkaline pH showed bands of polymerized myosin heavy chain on SDS-PAGE pattern. The transition temperatures of recovered protein were 33.1, 44.3 and 65.5$^{\circ}C$. Gelation of recovered protein from alkali processing was estimated by increase of $\beta$-sheet structure by pH treatment, S-S bonds by oxidation of surface sulfhydryl group in heating, polymerization of myosin heavy chain in order.r.

• Korean Journal of Food Science and Technology
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• v.21 no.3
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• pp.364-369
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• 1989

Experiments were carried out to investigate on the effect of cryoprotectants to the quality changes of pork and beef muscles during frozen storage . Beef and pork muscles were mixed with various cryoprotectants and stored at $-20^{\circ}C$ in a chest freezer for 12 weeks. Samples were analyzed for pH changes, TBA value, free atty acid contents, water and salt soluble protein extractability. The results obtained are summerized as follows. The pH value in all of cryoprotectants added samples were increased up to 0.25-0.5 as in non-treated samples . The TBA value, free fatty acid contents were increased with storage time as compared with the non-treated sample. Cryoprotectant effect on water soluble protein extractability was greater in pork than in beef muscle during frozen storage, especially in pork muscle treated CP-B, mixture of sorbitol, sucrose and sodium tripolyphosphate, as compared with non-treated sample. Cryoprotectant effect on salt soluble protein extractability during frozen storage was more pronounced in the beef muscle treated with CP-A which was mixture of sorbitol, mono sodium glutamate and sodium tripolyphosphate, and in the pork muscle treated with CP-B, mixture of sorbitol, sucrose and sodium tripolyphosphate than in the non-treated sample.

• Korean Journal of Food Science and Technology
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• v.21 no.2
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• pp.173-179
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• 1989

The influence of the storage temperature and time after slaughter on the thermal denaturation of PSE porcine muscle protein was studied by differential scanning calorimetry and by measuring the solubility of the sarcoplasmic proteins. In the DSC therodiagram a decrease of the endotherm enthalpy of the myosin plus sarcoplasmic proteins in PSE muscle could be observed with an increase in the storage temperature and time of post mortem. Storage temperature at $20^{\circ}C$ during the first four hours of post mortem resulted in relatively slight denaturation of myosin plus sarcoplasmic proteins in PSE muscle. Storage temperature above $25^{\circ}C$ caused to increase the denaturation of muscle proteins. The minimal drip loss in PSE muscle could be observed, when the muscle was cooled to $2^{\circ}C$ as quickly as possible post mortem. However, when stored for several hours of post morte at a temperature between $32^{\circ}C-38^{\circ}C$, the drip loss reached the level established for PSE muscle. The paleness of PSE muscle could be prevented to some extent by rapid chill to $20^{\circ}C$ post mortem. The more the muscle proteins in the PSE muscle become denatured during the early storage period of post mortem, the more the drip loss increases. With the increase in the denaturation of myosin plus sarcoplasmic proteins in PSE muscle with regard to temperature of post mortem, there was a corresponding decrease in the solubility of the sarcoplasmic proteins in PSE muscle.

• 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.