Department of Bioengineering, Stanford University, Stanford, CA, USA.
Biomacromolecules. 2011 Jun 13;12(6):2275-84. doi: 10.1021/bm200358r. Epub 2011 May 24.
Protein-based polymers are increasingly being used in biomaterial applications because of their ease of customization and potential monodispersity. These advantages make protein polymers excellent candidates for bioanalytical applications. Here we describe improved methods for producing drag-tags for free-solution conjugate electrophoresis (FSCE). FSCE utilizes a pure, monodisperse recombinant protein, tethered end-on to a ssDNA molecule, to enable DNA size separation in aqueous buffer. FSCE also provides a highly sensitive method to evaluate the polydispersity of a protein drag-tag and thus its suitability for bioanalytical uses. This method is able to detect slight differences in drag-tag charge or mass. We have devised an improved cloning, expression, and purification strategy that enables us to generate, for the first time, a truly monodisperse 20 kDa protein polymer and a nearly monodisperse 38 kDa protein. These newly produced proteins can be used as drag-tags to enable longer read DNA sequencing by free-solution microchannel electrophoresis.
蛋白质聚合物由于其易于定制和潜在的单分散性，在生物材料应用中越来越受到重视。这些优势使得蛋白质聚合物成为生物分析应用的优秀候选者。在这里，我们描述了用于生产游离溶液共轭电泳（FSCE）的拖曳标签的改进方法。FSCE 利用纯的、单分散的重组蛋白，末端连接到 ssDNA 分子上，以在水性缓冲液中实现 DNA 大小分离。FSCE 还提供了一种高度灵敏的方法来评估蛋白质拖曳标签的多分散性，从而评估其在生物分析中的适用性。该方法能够检测拖曳标签电荷或质量的微小差异。我们设计了一种改进的克隆、表达和纯化策略，使我们能够首次生成真正单分散的 20 kDa 蛋白质聚合物和近单分散的 38 kDa 蛋白质。这些新生产的蛋白质可用作拖曳标签，通过游离溶液微通道电泳实现更长的读 DNA 测序。
