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Building the Outer Membrane Protein G (OmpG) Nanopore Library: From the Discrimination of Biotin-Binding Proteins in Serum to Resolving Human Carbonic Anhydrase From Human Red Blood Cells

Abstract
The use of pore-forming proteins (PFPs) in nanopore sensing has been fruitful largely due to their nanoscale size and the ease with which protein nanopores can be manipulated and consistently reproduced at a large scale. Nanopore sensing relies heavily on a steady ionic current afforded by rigid nanopores, as the change in current is indicative of analyte detection, revealing characteristics of the analyte such as its relative size, concentration, and charge, as well as the nanopore:analyte interaction. Rigid PFPs have been used in applications such as DNA sequencing, kinetic studies, analyte discrimination, and protein conformation dynamics at the single-molecule level. The work in this dissertation details a different approach to nanopore sensing using a non-rigid PFP, the monomeric outer membrane protein G (OmpG), which exhibits intrinsic current fluctuations called gating. We exploit the gating of OmpG for the effective detection and discrimination of protein homologues and isoforms, showing that OmpG gating is particularly useful in selectively detecting targets and probing nanopore:analyte interactions. Further, we demonstrate the ability of OmpG to retain its sensing capabilities in complex mixtures of serum and human cell lysate.
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