Oxidative refolding of proteins / 生物工程学报

ABSTRACT

The mechanism of oxidative refolding of proteins was elucidated in more detail from the intensive and extensive studies in the past decades. 1. Most of the proteins examined so far proceed oxidative refolding via multiple pathways rather than a single and specific pathway. This is consistent with the folding energy landscape theory. 2. It is the native interactions rather than the non-native interactions that direct the folding process. This is not necessarily incompatible with the importance of the non-native disulfide intermediates in the bovine pancreatic trypsin inhibitor (BPTI) pathway, which are just a chemical necessity in the intramolecular arrangement to facilitate native disulfide formation. 3. Based on the BPTI refolding it was suggested that disulfide bonds have a stabilizing effect on the native state without determining either the folding pathway or the final three-dimensional structure of the protein. This point of view is not applicable to other proteins. Studies on the refolding of prochymosin unequivocally demonstrated that the formation of native disulfides is the prerequisite to the recovery of the native conformation. It is more likely that the interdependence between the native disulfide formation and the formation of native structure is a general rule. 4. At the early stage of oxidative refolding disulfide formation is essentially a random process, with the progress of refolding further disulfide formation is increasingly dependent on the conformations of the intermediates. Enhancing the renaturation yield of recombinant proteins is a major challenge in biotechnology. In addition to aggregation, the formation of species with mispaired disulfide bonds is a leading cause of decreased yield. Progress in understanding the mechanism of oxidative refolding has provided insight into how to solve this problem. As described above, at the later stage of refolding disulfide formation depends on the conformations of intermediates. The intermediates with native-like and flexible structure favourable for native disulfide formation and correct refolding are productive intermediates, while the unproductive intermediates tend to adopt stable conformations, which render the thiol groups and disulfide bond(s) inaccessible and further folding unfavourable energetically. Therefore, the principle to enhance the renaturation yield of disulfide-containing proteins is to cause the productive intermediates to predominate by destabilizing the unproductive intermediates. To approach this, alkaline pH, low temperature, labilizing agents, protein disulfide isomerase and its analogues and alteration of primary structure have been proved useful to adjusting the structure of the unproductive intermediates so as to facilitate thiol/disulfide interchange and in turn the native disulfide formation. The prospects for the oxidative refolding of proteins both in basic and applied researches are discussed in this review article.