Biochemistry & Molecular Biology
Characterization of Enzyme Activity and Substrate Specificity of LipW, a Lipase Linked to Tuberculosis
Document Type
Poster Presentation
Location
Indianapolis, IN
Subject Area
Biochemistry & Molecular Biology
Start Date
11-4-2014 8:30 AM
End Date
11-4-2014 9:30 AM
Sponsor
Jeremy Johnson (Butler University)
Description
Francisella tularensis is a gram-negative bacterium and the causative agent of tularemia, a highly deadly human disease. In this project, I worked to mutate five amino acids in a flexible loop of an important bacterial carboxylesterase (FTT258) from F. tularensis and to combine these five mutations with other key amino acids in FTT258. This flexible loop in FTT258 binds to bacterial membranes and was proposed to control the enzymatic activity of FTT258. All five amino acids were mutated using the QuikChange mutagenesis protocol. Some mutagenesis reactions, including the reaction for inserting D68A FTT258, required substantial optimization of the thermal conditions for the PCR reaction to obtain a successful mutation. After mutagenesis, all of the protein variants were purified by Ni-metal affinity chromatography and correct purification confirmed by SDS-PAGE. In ongoing research, the protein variants of FTT258 will now be used to assign the roles of each amino acid in the membrane binding and enzymatic activity of FTT258.
Characterization of Enzyme Activity and Substrate Specificity of LipW, a Lipase Linked to Tuberculosis
Indianapolis, IN
Francisella tularensis is a gram-negative bacterium and the causative agent of tularemia, a highly deadly human disease. In this project, I worked to mutate five amino acids in a flexible loop of an important bacterial carboxylesterase (FTT258) from F. tularensis and to combine these five mutations with other key amino acids in FTT258. This flexible loop in FTT258 binds to bacterial membranes and was proposed to control the enzymatic activity of FTT258. All five amino acids were mutated using the QuikChange mutagenesis protocol. Some mutagenesis reactions, including the reaction for inserting D68A FTT258, required substantial optimization of the thermal conditions for the PCR reaction to obtain a successful mutation. After mutagenesis, all of the protein variants were purified by Ni-metal affinity chromatography and correct purification confirmed by SDS-PAGE. In ongoing research, the protein variants of FTT258 will now be used to assign the roles of each amino acid in the membrane binding and enzymatic activity of FTT258.