Monodisperse, "highly" positively charged protein polymer drag-tags generated in an intein-mediated purification system used in free-solution electrophoretic separations of DNA

Biomacromolecules. 2012 Jan 9;13(1):117-23. doi: 10.1021/bm2013313. Epub 2011 Dec 23.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • DNA / chemistry*
  • DNA / genetics
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / genetics
  • Electrophoresis / methods
  • Inteins*
  • Sequence Analysis, DNA / methods*

Substances

  • DNA-Binding Proteins
  • DNA