Extracellular expression, oxidation and purification of hen egg white lysozyme double mutant (H15S+N77H)

Abstract

Metal catalyzed oxidation may result in structural damage to proteins and has been implicated in aging and disease, including neurological disorders like Alzheimer's. The selective modification of specific amino acid residues with high metal ion affinity leads to subtle structural changes that are not easy to detect and may have dramatic consequences on physical and functional properties of the oxidized protein molecule. This thesis is about the investigation of the site specificity of metal catalyzed oxidation of the H15S+N77H double mutant of hen egg white lysozyme. An extracellular mutant was generated using the PCR overlap extension method. The mutant was cloned into the pCR® 4-TOPO® vector. Sequencing showed the successful generation of an H15S+N77H double mutant capable of extracellular expression into pPICZB [alpha] B vector. The double mutant gene insert was digested out of pCR® 4-TOPO® vector and ligated in the pPICZ [alpha] B vector. Future work includes the linearization of pPICZ [alpha] B and transformation into X-33 competent cells, followed by the extracellular expression of mg quantities of the mutant protein. The results of metal catalyzed oxidation experiments will be compared to those of the native enzyme to reach conclusions about the relationships between site-specific oxidation and protein structure.