| Research efforts are described towards the creation of a novel series of non-natural, repetitive polypeptides (“protein polymers”) expressed in bacteria, which are designed to serve as “drag-tags” for microchannel DNA separation by End-Labeled Free-Solution Electrophoresis (ELFSE). ELFSE is a promising bioconjugate method for DNA sequencing and genotyping by both capillary and microfluidic device electrophoresis, which will eliminate the need for loading viscous polymer networks into electrophoresis microchannels. To accomplish microchannel DNA separations with high performance, ELFSE requires totally monodisperse perturbing entities (i.e. drag-tags), such as proteins, that will create a large amount of frictional drag when pulled behind DNA during free-solution electrophoresis, and which have other properties suitable for microchannel electrophoresis. Natural proteins are unsuitable drag-tags due to their compact folded shapes, surface charges, and strong interaction with the walls of the electrophoresis microchannel. However, non-natural, repetitive polypeptides hold substantial promise as ELFSE drag-tags because their sequences can be designed for desired properties, such as water-solubility and charge-neutrality. A novel controlled cloning method, developed in the course of the Ph.D. research, has enabled us to produce a series of long protein polymers (up to 1033 amino acids in length) having non-natural sequences of interest. This novel method allows production of long synthetic genes in a controlled and reproducible fashion, without intra-molecular cyclization. The method, which should be broadly applicable, was used to create long concatamer genes for subsequent cloning. The proteins were expressed, purified, and characterized by analytical HPLC, mass spectrometry, NMR, UV/VIS spectrometer, and circular dichroism (CD) spectroscopy. Through these characterization methods, it was determined that the protein polymers we obtained have many of the desired biophysical properties, including virtual monodispersity, the correct molecular weight, correct amino acid composition, high hydrophilicity, and a disordered, random-coil conformation. The resulting proteins were then conjugated to ssDNA oligomers using sulfo-SMCC as a coupling reagent and tested the performance of drag-tags in free-solution capillary electrophoresis. Based on microchannel electrophoretic analyses, we have demonstrated that high-resolution separation of DNA can be implemented by conjugating these polypeptide drag-tags to DNA. |
