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

我们的目的是阐明短S-S环在氧化蛋白重折叠中的作用。在本研究中，我们采用了具有两个短S-S环的重组人白细胞介素6 （rhIL-6）。还原后的rhIL-6在含有不同浓度的变性剂的溶液中再氧化，并检查了正确的二硫键合的rhIL-6的产率。即使存在高浓度的变性剂（6M氯化胍或9M尿素），rhIL-6也几乎被正确氧化。然而，化学反应的选择性不可避免地降低了，氧化过程中复杂副产物的形成是一个问题，虽然副产物的数量很少。另一方面，当rhIL-6在三级结构形成后被氧化时，选择性得到了提高。但rhIL-6容易聚集，特别是在高蛋白质浓度下，因此需要一定的装置来抑制聚集。我们测试了各种添加剂（包括几种试剂的组合）对rhIL-6的再折叠的影响，并成功地在0.5mg/ml蛋白质浓度下，使用1M尿素和1M LiCl将收率提高到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再折叠介质，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各有优点和缺点，但应该强调的是，之前的氧化简化了再折叠过程，有利于大规模工业生产。此外，由于S-S环的形成，即使是较短的S-S环在高变性浓度下也可能形成，稳定了rhIL-6的三级结构，促进了其再折叠，因此通过重组DNA技术将这种较短的S-S环引入难以再折叠的蛋白质中可能是提高其再折叠率的有效策略。

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.