Intracellular expression of the H15S hen egg white lysozyme mutant and the generation of the extracellular H15S mutant and transformation into Pichia pastoris

Abstract

Systems utilizing transition metal ions such as iron and copper are responsible for the production of free radicals such as hydroxyl ions via the Fenton reaction. Free radicals and the resulting oxidative damage have been shown to be involved in over 100 human diseases. This thesis is about the investigation of the site specificity of metal catalyzed oxidation of the H15S site directed mutant of hen egg white lysozyme. Histidine at position 15 was converted to serine by site directed mutagenesis, the mutant was then cloned into pCR4-TOPO. Top 10 E. coli was then transformed with the H15S/pCR4-TOPO plasmid. The mutant gene was then ligated into the yeast expression vector, pPICZB, designed for intracellular expression, and pPICXα A designed for the extracellular expression of protein in the methylotropic yeast, Pichia pastoris. The intracellular expression of protein was carried with three different types of media. Large scale expression was carried out in BMMH media, as this media gave the best evidence for the small scale expression of the H15S mutant lysozyme. Faint bands were seen for BMMH with SDS-PAGE, but no protein was detected with an enzyme assay or the ELISA. An extracellular mutant was generated using the PCR overlap extension method. The mutant was cloned into the pCR4-TOPO vector. Sequencing showed the successful generation of an H15S lysozyme mutant capable of extracellular expression in a pPICZα plasmid. The H15S insert was digested out of pCR4-TOPO and ligated into the pPICZα A vector. The pPICZα A was linearized and transformed into X-33 competent cells. Future work requires the expression of mg quantities of the mutant protein. This will be followed by metal catalyzed oxidation experiments. Finally the oxidation pattern of the H15S mutant will be compared to that of the native lysozyme.