Transformation of the X-33 strain of Pichia pastoris and the small scale expression of the N103H mutant hen egg white lysozyme gene

Abstract

Reactive oxygen species (ROS) are molecules or radicals which are produced from oxygen. Most ROS are highly reactive due to the presence of unpaired electrons in their valence shell. Metal ions such as Fe²⁺ or Cu⁺ are used as reducing agents in metal catalyzed oxidation (MCO) systems that generate the hydroxyl radical in a reaction with hydrogen peroxide. To study the oxidation of proteins by MCO systems a N103H mutant of hen egg white lysozyme (HEWL) was generated. The HEWL mutant N103H gene was successfully cloned into the pPICZÜA plasmid and subsequently transformed into Mach1[superscript TM]-T1[superscript R] chemically competent Escherichia coli. The recombinant plasmid was isolated, linearized, and transformed into the X-33 strain of the yeast Pichia pastoris by electroporation. Small scale expression was performed using a buffered glycerol media and methanol media at pH 6.0 and 7.0 for 4 days. Expression in unbuffered media was also performed. SDS-PAGE analysis of the supernatant samples from small scale expression revealed extracellular expression of a protein of the right size in a buffered medium whereas unbuffered medium showed no evidence of the expression of any proteins. Small scale expression in buffered media at pH 7.0 with antifoam appeared to give the best protein expression. A Bradford assay indicated the extracellular expression of protein to a concentration of about 0.1 mg/mL. Enzyme assays of pooled and concentrated fractions collected over four days showed no lysozyme activity. A low concentration of the N103H HEWL mutant might be the reason for no activity.