화학공학소재연구정보센터
Biomacromolecules, Vol.13, No.1, 117-123, 2012
Monodisperse, "Highly" Positively Charged Protein Polymer Drag-Tags Generated in an Intein-Mediated Purification System Used in Free-Solution Electrophoretic Separations of DNA
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 similar to 400 bases read length possible when more "highly" positively charged protein polymers of larger size are generated with the intein system.