The Role of Short S-S Loop : Can it be the Core for Refolding of Globular Proteins?

短S-S环的作用：它可以成为球状蛋白重折叠的核心吗？

• 批准号: 06672149
• 负责人: SAKAI Tomoya
• 金额: $ 1.22万
• 依托单位: Nagoya City University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06672149/
• 关键词: 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

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.

我们的目标是阐明短S-S环在氧化蛋白质重折叠中的作用。本研究采用具有两个短S-S环的重组人白细胞介素6（rhIL-6）。还原的rhIL-6在含有不同浓度变性剂的溶液中被再氧化，并检测了正确的二硫键结合的rhIL-6的产率，即使在高浓度变性剂（6 M盐酸胍或9 M尿素）存在下，rhIL-6也几乎被正确地氧化。然而，化学反应的选择性不可避免地降低，并且在氧化过程中形成复杂的副产物是一个问题，尽管副产物的量很少。另一方面，当rhIL-6在三级结构形成后被氧化时，选择性提高。但rhIL-6在高蛋白浓度下易聚集，因此需要一定的装置来抑制聚集。我们用1 M尿素和1 M氯化锂作复性剂，在蛋白质浓度为0.5mg/ml时，成功地将rhIL-6的复性率提高到90%。然而，在其他蛋白质的情况下，有必要为每种蛋白质设计最佳的重折叠介质，这并不总是容易的。因此，在三级结构形成之前或之后的氧化分别具有优点和缺点，但应该强调的是，之前的氧化简化了复性过程，并且有利于大规模工业生产。此外，由于S-S环的形成，即使是在高变性剂浓度下可能形成的短S-S环，也稳定了rhIL-6的三级结构并促进了其重折叠，因此通过重组DNA技术将这种短S-S环引入难以重折叠的蛋白质可能是提高其重折叠产率的有效策略。