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Publication Date

2015-07-04

Availability

UM campus only

Embargo Period

2017-07-04

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Chemistry (Arts and Sciences)

Date of Defense

2015-06-12

First Committee Member

Leonidas Bachas

Second Committee Member

Roger Leblanc

Third Committee Member

James Wilson

Fourth Committee Member

Sapna Deo

Abstract

Genetic engineering has provided us with the ability to advance medicine and technology by creating proteins that can be used for various applications. Two such applications are analyte detection and energy production. As more knowledge about protein structure and function becomes available and modeling programs are improved, researchers become better equipped to design and create mutant proteins to fit desired needs. In this research, we have genetically engineered proteins to create mutants that can be used for detection of analytes of interests and/or in biofuel cell development. The bioluminescent protein aequorin (AEQ) was mutated in a way that allows the protein to detect the protease caspase-3, a marker for apoptosis, by creating a measurable change in bioluminescence in the presence of the protease. We characterized the mutant and compared it to AEQ that had not been engineered to detect caspase-3. Then, we used this mutant AEQ to detect apoptosis both in vitro and in vivo. This dissertation also describes a mutated redox enzyme, glucose oxidase (GOx). Glucose oxidase has both the ability to detect glucose and to utilize glucose for energy at the anode of a biofuel cell. We engineered GOx to create a mutant that has the ability to transfer electrons more efficiently to an electrode, than the wild type enzyme. This was accomplished by mutating the amino acid that is the potential site of electron transfer from the enzyme's active center to oxygen. Finally, we expressed and purified two thermophilic proteins, phenol hydroxylase and laccase, that are both desired because of their stability and potential use at the cathode of biofuel cells.

Keywords

protein engineering; apoptosis; cornea; caspase-3; aequorin; glucose oxidase

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