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

我们的目标是阐明S-S短环在氧化蛋白质折叠中的作用。在本研究中，我们采用了具有两个S-S环的重组人白细胞介素6(rh IL-6)。在含有不同浓度变性剂的溶液中，还原的rhIL-6被重新氧化，并检测了正确结合二硫键的rhIL-6的产率。即使在高浓度变性剂(6M氯化胍或9M尿素)的存在下，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了，虽然副产物的量很少，但在氧化过程中形成复杂的副产物是一个问题。另一方面，当rh IL-6在形成三级结构后被氧化时，选择性得到提高。但rhIL-6容易聚集，特别是在高蛋白浓度下，因此需要某些装置来抑制聚集。我们考察了多种添加剂的组合对重组人IL-6复性的影响，在0.5 mg/ml的蛋白质浓度下，1M尿素和1M LiCl的加入量可使产量提高到90%左右。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复性介质，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各有优缺点，但应强调的是，先前的氧化简化了折叠过程，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环，也稳定了重组人IL-6的三级结构，促进了其复性，因此，利用重组技术将这种短的S-S环引入到难以复性的蛋白质中，可能是提高其复性得率的有效策略。

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.