| Project/Area Number |
06672149
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Physical pharmacy
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| Research Institution | Nagoya City University |
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Principal Investigator |
SAKAI Tomoya Nagoya City University, Faculty of Pharmaceutical Science, Department of Chemical Reaction Engineering, Professor, 薬学部, 教授 (00080169)
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| Co-Investigator(Kenkyū-buntansha) |
KURIMOTO Eiji Nagoya City University, Faculty of Pharmaceutical Science, Department of Chemica, 薬学部, 助手 (90234575)
KURODA Yoshitaka Nagoya City University, Faculty of Pharmaceutical Science, Department of Chemica, 薬学部, 講師 (40080204)
NOHARA Daisuke Nagoya City University, Faculty of Pharmaceutical Science, Department of Chemica, 薬学部, 助教授 (60080214)
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| Project Period (FY) |
1994 – 1996
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| Project Status |
Completed (Fiscal Year 1996)
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| Budget Amount *help |
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1996: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1995: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1994: ¥600,000 (Direct Cost: ¥600,000)
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| Keywords | Disulfide bond / Short S-S loop / Protein Refolding / Protein Folding / Protein Structure / Glutathione / Cytokine / N-ループS-S結合 / 小ループS・S結合 |
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| Research Abstract |
Our objective is to clarify the role of short S-S loops in oxidative protein refolding. In this study, we adopted recombinant human interleukin 6 (rhIL-6) having two short S-S loops. Reduced rhIL-6 was reoxidized in solutions containing various concentrations of a denaturant and yields of correctly disulfide-bonded rhIL-6 were examined.
Even in the presence of high concentration of a denaturant (6M Guanidinium chloride or 9M urea), rhIL-6 was almost correctly oxidized. However, the selectivity of chemical reactions inevitably became lowered, and the formation of complex by-products during oxidation was a problem though the amount of the by-products was small. On the other hand, when rhIL-6 was oxidized after the formation of tertiary structure, the selectivity was improved. But rhIL-6 was prone to aggregate especially at high protein concentration, hence certain devices were required to repress aggregation. We tested the effects of various additives including a combination of several reagents on the refolding of rhIL-6 and succeeded to raise the yield up to about 90% at 0.5mg/ml protein concentration using 1M urea and 1M LiCl. In cases of other proteins, however, it is necessary to design the optimal refolding media for each protein, and that is not always easy. Thus, oxidation that precedes or succeeds the formation of tertiary structure has both merits and demerits, respectively, but it should be emphasized that preceding oxidation simplifies refolding process and is advantageous to large-scale industrial production. Furthermore, since the formation of S-S loop, even short one possible to form at high denaturant concentration, stabilized the tertiary structure of rhIL-6 and facilitated its refolding, it gives rise to the possibility that the introduction of such short S-S loops by recombinant DNA technology to a protein difficult to refold could be an effective strategy to improve its refolding yield.
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