Monodisperse, “Highly”
Positively Charged
Protein Polymer Drag-Tags Generated in an Intein-Mediated Purification
System Used in Free-Solution Electrophoretic Separations of DNA
posted on 2012-01-09, 00:00authored byXiaoxiao Wang, Jennifer
Coyne Albrecht, Jennifer S. Lin, Annelise E. Barron
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.