Removal of the Purification Tag in Putative Toxin Protein BC_2332 from Bacillus Cereus via Site-Specific Mutagenesis and Restriction Enzyme Cutting
Date of Award
Restricted Access Thesis
College of Theology, Arts, & Sciences
Math & Science
Dr. Michael Godsey
Putative toxin protein BC_2332, isolated from Bacillus cereus which is a relative of Bacillus anthracis, shows similarity in the active site to ADP-ribosylating proteins present in cholera and diptheria. Preliminary attempts to measure the activity of the putative toxin BC_2332 were unsuccessful leading to the hypothesis that the purification tag is hindering the activity. This hypothesis is supported by crystal structure analysis showing the putative active site occupied by the purification tag. My objective was to remove the purification tag in order to relieve potential inhibition. The method entailed using site-specific mutagenesis and restriction enzyme cutting in order to genetically modify the expression system to remove the purification tag. We believe, but cannot verify, that this yielded a modified plasmid and further analysis is underway. This method along with other research is making progress towards removal of the purification tag to allow for measurement of the activity of the putative toxin protein.