Protein polymer drag-tags for DNA separations by end-labeled free-solution electrophoresis
Protein polymer drag-tags for DNA separations by end-labeled free-solution electrophoresis
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DOI：
10.1002/elps.200410042
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发表时间：
2005-06-01
期刊：
影响因子：
2.9
通讯作者：
Barron, AE
中科院分区：
文献类型：
--
作者：
Won, JI;Meagher, RJ;Barron, AE
我们论证了使用基因工程蛋白聚合物作为拖拽标签进行末端标记自由溶液电泳(ELFSE)分离DNA的可行性。蛋白质聚合物是极有前途的ELFSE拖拽标签候选者，因为它们的序列和长度是可控的，不仅可以产生具有高摩擦阻力的单分散聚合物，而且还可以满足通过微通道电泳进行高分辨率分离的其他拖拽标签要求。设计了一系列重复的多肽，在大肠杆菌中进行了表达和纯化。通过将蛋白质聚合物与荧光标记的DNA低聚物(22个碱基)进行端接偶联，并用自由溶液毛细管电泳法(CE)分析偶联分子的电泳迁移率，考察了蛋白质聚合物拖拽标签的大小和电荷的影响。此外，通过将荧光标记的聚合酶链式反应(PCR)产物偶联到带电荷的中性蛋白质聚合物上，并通过CE分析，观察了由相对较长的DNA片段(108和208个碱基)组成的附着在不带电荷的拖拽标签上的生物偶联物的电泳行为。我们计算了各种阻力标签产生的摩擦力，并估计了如果这些阻力标签用于DNA测序，在我们目前的系统中可以获得的潜在阅读长度。这些研究的结果表明，较大且不带电荷的拖拽标签将具有最佳的DNA分辨能力用于ELFSE分离，理论上，使用我们生产的一种蛋白质聚合物拖拽标签可以对多达233个DNA碱基进行测序，这种拖拽标签是静电中性的，链长度为337个氨基酸。我们还表明，变性(未折叠)多肽链比折叠蛋白(如链霉亲和素)在单位分子量上施加的摩擦阻力要大得多，链霉亲和素以前曾被用作拖拽标签。
We demonstrate the feasibility of end-labeled free-solution electrophoresis (ELFSE) separation of DNA using genetically engineered protein polymers as drag-tags. Protein polymers are promising candidates for ELFSE drag-tags because their sequences and lengths are controllable not only to generate monodisperse polymers with high frictional drag, but also to meet other drag-tag requirements for high-resolution separations by microchannel electrophoresis. A series of repetitive polypeptides was designed, expressed in Escherichia coli, and purified. By performing an end-on conjugation of the protein polymers to a fluorescently labeled DNA oligomer (22 bases) and analyzing the electrophoretic mobilities of the conjugate molecules by free-solution capillary electrophoresis (CE), effects of the size and charge of the protein polymer drag-tags were investigated. In addition, the electrophoretic behavior of bioconjugates comprising relatively long DNA fragments (108 and 208 bases) and attached to uncharged drag-tags was observed, by conjugating fluorescently labeled polymerase chain reaction (PCR) products to charge-neutral protein polymers, and analyzing via CE. We calculated the amount of friction generated by the various drag-tags, and estimated the potential read-lengths that could be obtained if these drag-tags were used for DNA sequencing in our current system. The results of these studies indicate that larger and uncharged drag-tags will have the best DNA-resolving capability for ELFSE separations, and that theoretically, up to 233 DNA bases could be sequenced using one of the protein polymer drag-tags we produced, which is electrostatically neutral with a chain length of 337 amino acids. We also show that denatured (unfolded) polypeptide chains impose much greater frictional drag per unit molecular weight than folded proteins, such as streptavidin, which has been used as a drag-tag before.