Sharma L, Sharma A
Science Department, Davenport University, Midland, MI, USA.
Eur J Biochem. 2001 Apr;268(8):2456-63. doi: 10.1046/j.1432-1327.2001.02125.x.
A common obstacle to proper renaturation of an unfolded protein is aggregation, an intermolecular side reaction of immense importance in biotechnology and in the pathogenesis of several neurodegenerative diseases. Cyclic sugars known as cyclodextrins have been used as protein-folding aids. The effect of cyclodextrin chemistry on aggregation and refolding of carbonic anhydrase was evaluated in this study. Size-exclusion HPLC showed that cyclodextrins inhibit aggregate formation without interfering with the correct renaturation of carbonic anhydrase. PAGE of refolded enzyme provides further evidence of inhibition of folding-related aggregation by natural and chemically modified cyclodextrins. Although the amount of aggregate formed and recovery of active enzyme was dependent on cavity size, the nature of the chemical substituents found on the rims of the sugar molecule seems to play a more important role in cyclodextrin-assisted refolding of carbonic anhydrase. In general, neutral or cationic cyclodextrins with small cavities were found to be better folding aids than anionic cyclodextrins with larger holes. Although the exact prediction of the effect of a cyclodextrin substitution on protein refolding is not possible at present, these results clearly show that modified cyclodextrins can be designed that effectively inhibit protein aggregation.
未折叠蛋白正确复性的一个常见障碍是聚集，这是一种在生物技术和几种神经退行性疾病发病机制中极为重要的分子间副反应。被称为环糊精的环状糖类已被用作蛋白质折叠辅助剂。本研究评估了环糊精化学对碳酸酐酶聚集和复性的影响。尺寸排阻高效液相色谱表明，环糊精可抑制聚集体形成，而不干扰碳酸酐酶的正确复性。对复性酶进行的聚丙烯酰胺凝胶电泳进一步证明了天然和化学修饰的环糊精对与折叠相关的聚集的抑制作用。虽然形成的聚集体数量和活性酶的回收率取决于空腔大小，但糖分子边缘上化学取代基的性质似乎在环糊精辅助碳酸酐酶复性中起更重要的作用。一般来说，发现具有小空腔的中性或阳离子环糊精比具有较大孔洞的阴离子环糊精是更好的折叠辅助剂。虽然目前还无法准确预测环糊精取代对蛋白质复性的影响，但这些结果清楚地表明，可以设计出能有效抑制蛋白质聚集的修饰环糊精。
