Monodisperse, "highly" positively charged protein polymer drag-tags generated in an intein-mediated purification system used in free-solution electrophoretic separations of DNA.
Monodisperse, "highly" positively charged protein polymer drag-tags generated in an intein-mediated purification system used in free-solution electrophoretic separations of DNA.
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DOI：
10.1021/bm2013313
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发表时间：
2012-01-09
影响因子：
6.2
通讯作者：
Barron, Annelise E.
中科院分区：
文献类型：
--
作者：
Wang, Xiaoxiao;Albrecht, Jennifer Coyne;Lin, Jennifer S.;Barron, Annelise E.
Free-solution conjugate electrophoresis (FSCE) is a method of DNA sequencing that eliminates the need for viscous polymer solutions by tethering a carefully designed, mobility modifying “drag-tag” to each DNA molecule to achieve size-based separations of DNA. The most successful drag-tags to date are genetically engineered, highly repetitive polypeptides (“protein polymers”) that are designed to be large, water-soluble, and completely monodisperse. Positively charged arginines were deliberately introduced at regular intervals into the amino acid sequence to increase the hydrodynamic drag without increasing drag-tag length. Additionally, a one-step purification method that combines affinity chromatography and on-column tag cleavage was devised to achieve the required drag-tag monodispersity. Sequencing with a read length of approximately 180 bases was successfully achieved with a known sequence in free-solution electrophoresis using one of these positively charged drag-tags. This preliminary result is expected to lead to further progress in FSCE sequencing with ~400 bases read length possible when more “highly” positively charged protein polymers of larger size are generated with the intein system.
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