Characterization and purification of bacteriophages using chromatofocusing
Characterization and purification of bacteriophages using chromatofocusing
复制标题
DOI：
10.1016/j.chroma.2008.08.037
复制
发表时间：
2008-10-17
影响因子：
4.1
通讯作者：
Frey, Douglas D.
中科院分区：
文献类型：
--
作者：
Brorson, Kurt;Shen, Hong;Frey, Douglas D.
将色谱聚焦技术应用于常规用于测试病毒过滤器的三种噬菌体的表征和纯化：phi X174、PR 772和PP 7。使用化学上明确定义的洗脱液缓冲液，而不是更常用的色谱聚焦聚两性电解质缓冲液。选择色谱柱填充物，以通过将噬菌体吸附仅限于外部颗粒表面来最小化条带增宽。在所使用的条件下，确定可以使噬菌体在保留的pH梯度中聚焦成窄带，其中活噬菌体的回收率范围为15%至接近100%，如通过噬斑形成测定所确定的。保留时间和表观等电点数据，获得的样品组成的纯化的噬菌体，或样品组成的噬菌体含有宿主细胞杂质的粗制备物。使用修改的先前描述的模型获得等电点估计值。所获得的结果表明，层析聚焦是一种简单而快速的方法，用于获得噬菌体和可能其他类型的病毒的近似等电点。这也可能是一种有用的方法来纯化这些材料。(C)2008 Elsevier B.V.保留所有权利。
The technique of chromatofocusing was applied to the characterization and purification of three bacteriophages that are routinely used for testing virus filters: phi X174, PR772, and PP7. Chemically well-defined eluent buffers were used, instead of the more commonly used chromatofocusing polyampholyte buffers. Chromatographic column packings were selected to minimize band broadening by confining bacteriophage adsorption solely to the exterior particle surface. Under the conditions used it was determined that bacteriophages could be made to focus into narrow bands in a retained pH gradient with recoveries of live phage that ranged from 15 to nearly 100% as determined by a plaque-forming assay. Retention times and apparent isoelectric point data were obtained for samples consisting either of purified bacteriophage, or samples consisting of crude preparations of bacteriophages containing host cell impurities. Isoelectric point estimates were obtained using modified, previously described models. The results obtained suggest that chromatofocusing is a simple and rapid method for obtaining approximate isoelectric points for bacteriophages and probably other types of viruses. It is also likely a useful method for purifying these materials. (C) 2008 Elsevier B.V. All rights reserved.