Characterization of a Hypothetical Hydrolase from Mycobacterium tuberculosis

Jakob Jozwiakowski Butler University
Faculty Sponsor(s): Randal Jeremy Johnson Butler University
The pathogen Mycobacterium tuberculosis is able to enter a state of dormancy in which the central metabolism shuts down. In this state, the pathogen is resistant to drug treatment and difficult to identify in host organisms. The hypothetical hydrolase Rv0272c is a potentially key lipid metabolism enzyme controlling this dormant state transition from Mycobacterium tuberculosis. Herein, we cloned the Rv0272c protein, expressed the protein in Mycobacterium smegmatis, and characterized the detailed substrate specificity of Rv0272c. Initially, the Rv0272c gene was cloned from the H37Rv Gateway cloning library into an expression vector with a built in C-terminal stop codon. Optimal conditions for Rv0272c expression were then determined using variable protein expression conditions (time, temperature, and induction agent). Active Rv0272c protein was then isolated by Nickel-affinity chromatography and confirmed by SDS-PAGE. Thermal stability reinforced that Rv0272c was fully folded and stable at room temperature. Assays against a substrate library determined the specificity of Rv0272c.
Biochemistry & Molecular Biology
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