Date of Award
Master of Science
Dr. Brian Tripp
Dr. John Geiser
Dr. Todd Barkman
Flagellin, carbonic anhydrase, protein engineering, zinc, salmonella
Masters Thesis-Open Access
Flagellin is the protein monomer that comprises the bacterial flagella for most bacteria, including Salmonella typhimurium. This protein has attracted attention for protein engineering because it is exported out of the cell, polymerized into stable fibers, is produced in large quantities and is relatively simple to purify. Using rational design with computer modeling, potential active sites in the flagellin protein structure were modeled after the human carbonic anhydrase II tetrahedral zinc binding site. In total, three locations were selected as potential active sites and the necessary mutations were successfully introduced. Flagella formation for the flagellin variants was demonstrated through TEM microscopy. When analyzed for metal ion content bound by the flagellin variants by ICP-ES, it appears that two of the variants have some level of zinc (II) association. One of these flagellin variants has diminished motility in motility agar that can be rescued upon the addition of the metal ion chelator EDTA and is less resistant to trypsin digestion upon the removal of metal ions with EDTA. Human carbonic anhydrase II can hydrolyze 4-NPA, however the flagellin variants have no detectable esterase activity. These preliminary results suggest that a metal binding site has successfully been introduced into bacterial flagellin while still retaining the ability of the protein to export, assemble and function as a motility organelle.
Haase, Alexandra M., "Catalytic Engineering of the Flagellin Protein" (2012). Master's Theses. 27.