Cathepsin L1 is a cysteine protease that has been previously isolated and functionally expressed in Saccharomyces cerevisiae. It has the potential to be employed as a vaccine for liver-fluke disease in cattle and other ruminants. Production of this recombinant enzyme, which is secreted into the media from recombinant yeast, was studied initially in shake flask cultures and subsequently in 5L and 15L fermenters. In early studies, low productivity and especially variations in Cathepsin L1 production was a significant problem. A standard operating protocol (SOP) has been designed to consistently supply an optimum inoculum for large-scale fermentations. This SOP which involved 'blending' colonies for inoculum cultures in conjunction with sub-culturing starter flasks for two successive cycles of 48 hours, proved to be the most successful for consistently high levels of enzyme production during the ensuing fermentation. The pH and temperature optima are pH 6.5 and 30°C respectively for culturing the recombinant yeast to produce both both high biomass levels and high enzyme activity. Addition of casamino acids to the selective media or replacing it with complex YEPD resulted in poor plasmid stability and low Cathepsin L1 production. By supplementing the selective media with extra yeast nitrogen base, using a glucose concentration of 20g/L, enzyme activity increased by 3-4 fold and much higher levels of plasmid stability than observed in non-selective media were sustained. Enzyme activity of 0.74 units/mL were obtained in supplemented media compared to 0.19 units/mL in selective media. Investigations were performed on the constitutive behaviour of the ADH1 promoter, which controls the expression of Cathepsin L1 in this recombinant yeast strain. It revealed that enzyme production is repressed at high concentrations of glucose but gradually increases as glucose is utilised. Cathepsin L1 is still expressed during the ethanol consumption phase, albeit at a slower rate than during the latter stages of glucose consumption.