Protein polymer drag-tags for DNA separations by end-labeled free-solution electrophoresis.
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.
Won
Jong-In
JI
Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL 60208, USA.
Meagher
Robert J
RJ
Barron
Annelise E
AE
D004247:DNA / Q000302:isolation & purification*; D046209:Electrophoresis, Microchip / Q000379:methods*; D005456:Fluorescent Dyes; D008968:Molecular Conformation; D015335:Molecular Probes; D009841:Oligonucleotides / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D015202:Protein Engineering; D017422:Sequence Analysis, DNA
D005456:Fluorescent Dyes; D015335:Molecular Probes; D009841:Oligonucleotides; D010455:Peptides; D004247:DNA