Design and production of protein-based polymers for application as 'drag-tags' in free-solution DNA sequencing

End-Labeled Free-Solution Electrophoresis, or ELFSE, is an alternative strategy for DNA sequencing that proposes to eliminate the need for a viscous sieving matrix for size-based DNA separation. In this bioconjugate method, a perturbing entity or "drag-tag" is attached to differently sized DNA fragments produced by the Sanger reaction. This drag-tag alters the overall charge-to-friction ratio so that DNA separation by size can be accomplished by free-solution electrophoresis. Rapid separations with long read lengths are theoretically possible using ELFSE. Application of ELFSE to integrated "lab-on-a chip" microfluidic devices currently under development is facilitated by the absence of a viscous polymer solution.;For successful DNA sequencing, this perturbing entity needs to be large, water-soluble, preferably uncharged, monodisperse, and also have a point for unique attachment to DNA. A non-natural repetitive polypeptide, or "protein polymer", has the potential to meet these numerous requirements for optimal ELFSE performance. A small (127 amino acids) drag-tag was previously used to demonstrate that ELFSE sequencing is possible using a protein polymer as the drag-tag. Obtaining a sufficiently large and monodisperse protein polymer drag-tag has been the focus of this research and achieving this goal proved to be more challenging than originally expected.;Charged and uncharged protein polymers of varying lengths were evaluated for their potential as drag-tags. Sequences incorporating additional hydrophobic residues were also investigated as well as an alternative purification strategy using self-cleaving affinity tags. Proteins expressed with an N-terminal affinity tag were compared to those expressed with a C-terminal affinity tag to determine the best method for obtaining monodisperse protein polymers. The biophysical properties of these drag-tags were measured via spectroscopy. Extensive investigation into aspects of protein polymer design, production, and purification was required to finally produce a sufficiently monodisperse drag-tag of double the previous size, bringing us closer towards the goal of achieving long-read ELFSE sequencing.