Investigation of site-specific metal catalyzed oxidation of the lysozyme double mutant (H15S + N77H)

Abstract

Although oxidative damage to proteins by reactive oxygen species is significant in many diseases, there are many open questions regarding the site - specificity of this damage by reactive oxygen species. The highly reactive hydroxyl radical reacts with the amino acids at the metal binding site or with the residues in the vicinity of metal binding site. This thesis investigates the site specificity of metal-catalyzed oxidation of a mutant hen egg white lysozme using Cu+2 and hydrogen peroxide to generate the hydroxyl radical. The double mutant lysozyme gene was generated with Asn77 changed to His (N77H) and His15 changed to Ser (H15S) by site-directed mutagenesis. The lysozyme mutant gene was cloned into the yeast expression vector, pPICZB. The methyltropic yeast P. pastoris was transformed with the vector to express the mutant protein. Various culture conditions were used to optimize protein production. Methods like HPLC and gel chromatography were used in an attempt to separate the mutant lysozyme.