引用

霍乱毒素B亚基(CTB)如果其前导肽被大肠杆菌来源的一种肽取代，就会以其五聚体形式从大肠杆菌中分泌出来。然而，当分子突变或与外源肽融合时，五聚体的分泌通常会严重受损。因此，我们尝试从大肠杆菌的包涵体(IBs)中再生五聚体CTB。盐酸胍溶解的IBs的逐步透析主要产生可溶性的高分子质量(HMM)聚集物，只有一小部分的五聚体。评估了三种重组均匀五聚体分子的方法:(i)使用五聚体线圈融合伙伴，期望它作为组装核心;(ii)优化折叠过程中的蛋白质浓度;以及(iii)在重新折叠前消除污染物。线圈-线圈融合有一些效果，但仍然产生了大量的HMM聚集物。将蛋白质浓度从0.05 mg/mL改变到5mg/mL几乎没有效果。相反，在重新折叠前消除污染物具有稳健的效果，只有五聚体被再生，没有检测到HMM聚集。 Surprisingly, the protein concentration at refolding was up to 5mg/mL when the contaminants were removed, with no adverse effects on refolding. The regenerated pentamer was indistinguishable in its biochemical and immunological characteristics from CTB secreted from E. coli or choleragenoid from Vibrio cholerae. This study provides a simple but very efficient strategy for pentamerizing CTB with a highly homogeneous molecular conformation, with which it may be feasible to engineer CTB derivatives and CTB fusion antigens.