Disulfide Bond Forming Enzyme Activity via Lysozyme Assay

Kerstin E. Peterson
Dr. Cassidy Dobson, Faculty Mentor

Disulfide Bond Forming Enzyme (DBF) is a unique chaperone protein found in the hyperthermophile Sulfolobus solfataricus. DBF has the ability to chaperone disulfide bonds without the presence of cysteine, unlike any other disulfide chaperone, which implies a novel mechanism of action. DBF is overexpressed in E. coli, purified by immobilized metal affinity column chromatography, and analyzed via SDS-PAGE. DBF activity is determined by how well it can refold lysozyme. By measuring the activity of refolded lysozyme by UV-Vis spectroscopy, we can monitor the ability of DBF to correct disulfide bonds. To optimize the activity of DBF, several conditions such as the ratio of DBF to lysozyme and the stringency of reagents are tested. Initial experiments have shown all proteins and substrates are stable upon storage. Further testing will be performed to determine the appropriate ratio of reagents to optimize the assay.

Keywords: DBF, Proteins, Lysozyme

Topic(s):Biochemistry and Molecular Biology
Chemistry

Presentation Type: Oral Presentation

Session: TBA
Location: TBA
Time: TBA