Continuous Production of a Novel Lysozyme via Secretion from the Yeast, Pichia pastoris

We have cloned a cDNA for bovine lysozyme c2, a novel lysozyme characteristic of the cow stomach and expressed its protein product by secretion using its native signal sequence in the methylotrophic yeast, Pichia pastoris . A semi–continuous fermentation process used for production resulted in extremely high cell densities and product concentrations. During fed–batch fermentation, bovine lysozyme concentrations exceeded 550 mg/liter, and cell densities exceeded 120 g dry cells/liter. In continuous culture, bovine lysozyme and cell concentrations remained constant at approximately 350 mg/liter and 100 g dry cells/liter, respectively. The recombinant product is a single biologically active species that constitutes >80% of the protein in media from expressing cells; the signal sequence of the pre–lysozyme is accurately processed.