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Thursday, May 26, 2011

Culture of Yeast for the Production of Heterologous Proteins

This unit describes culture of the yeast strains Saccharomyces cerevisiae and Pichia pastoris for the production of foreign proteins. The protocols listed here for S. cerevisiae are for three widely used types of promoter: galactose-regulated (GAL1, GAL7, GAL10), glucose-repressible (e.g., ADH2), and constitutive glycolytic (e.g., PGK or GAPDH). Minor variations to each can be made depending on the selection system used. The P. pastoris expression system uses integrating vectors with the methanol-regulated AOX1 promoter and HIS4 selection marker; although transformants are stable, they are generally grown in minimal selective medium. Methods are described for small-scale S. cerevisiae and P. pastoris cultures and also for high-density fermentations with these yeasts. A simple feeding strategy based on calculated feed rates is provided for S. cerevisiae and yields cell densities of 10 to 30 g/liter. In contrast, with P. pastoris, basic fermenter equipment is used to obtain extremely high-density cultures (e.g., 130 g/liter). Finally, a Support Protocol describes small-scale preparation of protein extracts.

Link to Full Protocol: http://www.currentprotocols.com/protocol/ps0508