Method for producing completely monodisperse, highly repetitive polypeptides for use with Free-Solution Conjugate Electrophoresis and other bioanalytical applications

Researchers from Prof. Annelise Barron's laboratory have developed a novel method for purifying large, biologically produced protein polymers for DNA sequencing and genotyping. This technique is used to generate truly monodisperse, highly repetitive polypeptides that can be used, for instance, as DNA “drag-tags” in Free-Solution Conjugate Electrophoresis (“FSCE”). These large, protein-based polymers enable significantly longer sequencing reads than the smaller drag-tags to which the technology was previously restricted by purity problems. This technology is particularly applicable for DNA separations on microfluidic platforms because it facilitates exon-length sequencing in an aqueous solution, without the need for a viscous sieving matrix.

The general clone construction/protein design and accompanying purification protocols can be used for generating protein-based polymers for pharmaceutical, biomaterials, or other research applications where precise characterization and true monodispersity of a protein modifier is important.

This schematic of FSCE shows the monodisperse protein drag-tag (blue) conjugated to the DNA sequencing fragment (red) with fluorescent dye attached for detection (yellow star). Covalent attachment of the drag-tag to the DNA molecule allows for size-based separation in an electric field with no sieving matrix.

Stage of Research
The inventors have purified drag-tags as long as ~ 500 amino acids. They used a 267-amino acid drag tag in FSCE to read 265 bases of DNA by capillary electrophoresis in 30 minutes, with no sieving matrix.