Download Previous studies have nonspecifically attached a single protein to a

Mutagenesis, Overexpression, and Purification of DNase for Single Molecule Studies
Denise Der
Mentor: Gregory Weiss
Previous studies have nonspecifically attached a single protein to a carboxylatefunctionalized carbon nanotube. In this study, a single enzyme will be covalently and
site-specifically attached to a nanotube in a nanocircuit, and will be electronically
monitored in real-time. Part of the investigation was accomplished by mutagenesis
followed by overexpression and purification three different DNase E9 mutants. The latter
portion of this project involves attachment to the nanotube through cysteine chemistry
and visualization of changes in conductance between the various DNase mutants as it
hydrolyzes DNA. Thus far, the three DNase variants have been successfully expressed,
purified, and characterized. Because these mutants have been shown to be absolutely pure
and still remain active after refolding them, they are ready for site-specific attachment to
the functionalized carbon nanotube. Ultimately, attaching the enzyme to the nanotube and
visualizing its conductance will allow us to elucidate kinetic information of a single
protein.