Solubilization and stabilization of bacteriophage MS2 in organic solvents
Solubilization and stabilization of bacteriophage MS2 in organic solvents
复制标题
DOI：
10.1002/bit.21245
复制
发表时间：
2007-06-01
影响因子：
3.8
通讯作者：
Clark, Douglas S.
中科院分区：
文献类型：
--
作者：
Johnson, Harvey R.;Hooker, Jacob M.;Clark, Douglas S.
研究了几种用于在有机溶剂中溶解噬菌体MS2的技术。将MS2从水相中直接萃取到含有2 mM AOT的异辛烷中，这是一种经证实的用于许多蛋白质的有机增溶的方法，但没有成功。然而，通过直接加入含有500 mM AOT的有机溶剂溶解了MS 2的预干燥样品。作为一种替代方法，使用AOT在异辛烷中制备含有MS 2水溶液的反胶束，通过溶剂蒸发和共沸干燥进行脱水，并在所选溶剂中重新溶解。的结构和微环境的有机增溶MS2进行了研究，通过紫外吸收，所附的solvatochromatic染料，色氨酸荧光，和原子力显微镜，所有这些都有助于证据的一个完全组装的衣壳在有机溶剂中。用硬脂酸在氯仿中衍生化溶解的MS2，说明可以使用完全不溶于水的试剂在有机溶解的衣壳上进行生物缀合反应。此外，有机溶解的噬菌体在90 ° C加热20分钟后保持感染性，而在水性缓冲液中或用氮气干燥的噬菌体在热处理方案后是无活力的。扩大范围的可用的化学修饰和增强的热稳定性的有机溶解的衣壳预示着良好的存储稳定的疫苗的配方预测的反应或暴露于有机介质。
Several techniques were examined for the solubilization of bacteriophage MS2 in organic solvents. Direct extraction of the MS2 from an aqueous phase into isooctane containing 2 mM AOT, a proven approach for the organic solubilization of many proteins, was not successful. However, predried samples of MS2 were solubilized through the direct addition of organic solvents containing 500 mM AOT. As an alternative procedure, reverse micelles containing aqueous solutions of MS2 were prepared in isooctane using AOT, dehydrated through solvent evaporation and azeotropic drying, and resolubilized in a solvent of choice. The structure and microenvironment of organic-solubilized MS2 were investigated by UV absorbance, the fluorescence emission of an attached solvatochromatic dye, tryptophan fluorescence, and atomic force microscopy, all of which contributed evidence for a fully assembled capsid in the organic solvent. The solubilized MS2 was derivatized with stearic acid in chloroform, illustrating that bioconjugation reactions can be performed on organic-solubilized capsids using reagents that are completely insoluble in water. Furthermore, the organic-solubilized phage remained infectious after heating at 90 degrees C for 20 min, whereas phage in aqueous buffer or dried with nitrogen were nonviable following the heat treatment protocol. The extended range of available chemical modifications and the enhanced thermal stability of the organic-solubilized capsids bodes well for the formulation of storage-stable vaccines predicated on reactions in or exposure to organic media.