Generation, Cloning, and Small-Scale Expression of Site-Directed Mutants of Hen Egg White Lysozyme in Pichia Pastoris

Abstract

Reactive oxygen species (ROS) are compounds that are produced by the reduction of molecular oxygen; these species can generate free radicals. Metal-catalyzed oxidation systems are experimental systems that use a metal ion, such as Fe²⁺ or Cu⁺,hydrogen peroxide, and often a reducing agent to generate a free radical. The purpose of this research is to generate a suite of four site-directed mutants that were used for transformation into Pichia pastoris for small-scale expression of mutant lysozyme. The mutant lysozymes expressed will be used in future oxidation studies to determine the relationship between the site of oxidation and the level of protein structure. Two of the mutant genes, N103H and N77H, were created using the Polymerase Chain Reaction and the mutations were confirmed by gene sequencing. The genes were ligated into the pPICZα A plasmid, which supported replication in both bacterial and yeast systems. The N103H gene was successfully cloned into the yeast plasmid. Two previously prepared clones in pPICZα A, H15S and H15S+N77H, were transformed into Mach1[superscript TM]-T1[superscript R] chemically competent Escherichia coli. Microgram amounts of the cloned plasmids were generated, and the plasmids were linearized and then transformed into Pichia pastoris by electroporation. The phenotype of the yeast colonies was determined and small-scale expression experiments were performed. The proteins were analyzed by SDS-PAGE and by a turbidimetric assay. One overnight growth in YPD media containing Zeocin followed by a five-day growth in YPM media with an initial concentration of 2% methanol was determined to give the highest expression of a protein that retained lysozyme activity.