Date of Award
Campus Access Thesis
College of Theology, Arts, & Sciences
Math & Science
Dr. Michael Godsey
Proteins are linear molecules that consist of amino acids and can be folded into globular configurations, which are further defined as primary, secondary, tertiary, and quaternary structures. The formation of a protein significantly contributes to its functionality and the structural versatility gives rise to an assortment of functions that are vital to the life of cells and thus to the functioning of organisms. Proteomic research is continually attempting to determine the roles of all cellular proteins, as well as the interactions that these proteins experience. With the assistance of bioinformatics, the proteome sequences of thousands of proteins have been solved; however the number of hypothetical proteins of unknown structure and function is escalating.
One such protein is putative toxin protein BC_2332, isolated from Bacillus cereus, which is a close relative of the animal and human pathogen Bacillus anthracis and the insect pathogen Bacillus thuringiensis. This protein has been crystallized using X-ray crystallography and the structure analyzed, but it has an unknown function. Protein BC_2332 exhibits similarity in the active site to ADP-ribosylating proteins present in cholera and diphtheria toxins. Understanding the pathogenesis of putative toxin protein BC_2332 is important because of its active site similarities to cholera toxin and diphtheria toxin proteins.
Based on the crystal structure, it is believed that the histidine purification tag of bacillus cereus protein BC_2332 is blocking the active site of the protein, causing the initial attempts to measure the activity of the protein to be unsuccessful. So, the bacterial cells were grown, expressed and expressed and purified using Immobilized Metal Affinity Chromatography. Tobacco Etch Virus (TEV) protease was then used to cleave the purification tag from the protein. The validity of the TEV protease reaction was analyzed using SDS-PAGE electrophoresis. Because the gel was unable to definitively indicate the effectiveness of the TEV protease a metal affinity resin was added to the TEV protease reaction as a control (see the materials and methods section beginning on page 14 for further details). The initial removal difficulty of the purification tag could possibly be due to the sequestration of the TEV cutting site in the putative active site. Thus, more trials and problem solving, such as better construction of a positive control, is needed before this process is complete.
Fridlund, Joanna, "Removal of the Histidine Purification Tag of Putative Toxin Protein BC_2332 found in Bacillus Cereus, Related to Bacillus Anthracis, via Proteolytic Cleavage" (2011). Undergraduate Theses. 44.