Effect of Glycerol as a Crowding Agent on Lysis Solubility of Putative Toxin BC 2332 Found in Bacillus Cereus
Date of Award
Restricted Access Thesis
College of Theology, Arts, & Sciences
Math & Science
Dr. Michael Godsey
Proteins consist of a chain of amino acids that fold via a combination of hydrogen bonds, salt bridges, hydrophobic attractions, and separate chaperone proteins to form a functioning unit. The structure of a protein dictates the function within the cell it is translated into. The role of proteins within the cell is an active field of research that continues to grow as genetic sequencing elucidates the language of DNA. One example of a protein recently discovered in need of analysis is putative toxin protein BC_2332, isolated from Bacillus cereus, related to the animal and human pathogen Bacillus anthracis and Bacillus thuringensis (Helgason, 2000). This protein has been crystallized via x-ray crystallography and had its structure solved, but its enzymatic function is still unknown (Godsey e al., 2007). Protein BC_2332 contains sequence similarity to the ADP-ribosylation active site found in cholera and diphtheria toxins. Analysis of the activity of putative toxin protein BC_2332 is important due to its structural similarity to these known pathological toxins. In order to quantify the activity of the protein, it is necessary to perform a modification by removing the purification tag residing on it. However, previous attempts of protein modification have been met with difficulties (Fridlund, 2011). These difficulties regard a preferential lysis buffer for maximal protein solubility implicit for sufficient protein purification and cutting steps downstream of the methods. To this end, a bacterial culture was grown, expressed, and sonicated using a variety of buffers containing compounds discussed in the literature. It was discovered that the addition of glycerol in the role of a crowding agent resulted in a larger level of protein solubility. Further investigation of theoretical buffer composites may lead to the discovery of effective buffer compositions that can solvate larger amounts of BC_2332.