Protease-Deficient Strains 81 81 From: Methods in Molecular Biology, Vol. 103: Pichia Protocols Edited by: D. R. Higgins and J. M. Cregg © Humana Press Inc., Totowa, NJ 7 Generation of Protease-Deficient Strains and Their Use in Heterologous Protein Expression Martin A. G. Gleeson, Christopher E. White, David P. Meininger, and Elizabeth A. Komives 1. Introduction In optimizing expression of heterologous protein, the issue of proteolysis is often an important factor, since many peptides and proteins are susceptible to degradation by proteases produced in the host organism. In such cases even if the protein product is expressed at high levels, overall yield can be drastically reduced through proteolysis, which not only reduces the amount of intact material but also complicates the recovery process. The use of protease-defi- cient strains has been shown to be a successful approach to improve the yield of fully active, expressed proteins in both Saccharomyces cerevisiae and Escherichia coli (1,2). Proteolysis can occur during expression or during the first stages of purification. Certainly, expression of intracellular proteins, which requires cell lysis for purification, will result in their exposure to proteases. Similarly, secreted proteins will be exposed to vacuolar proteases through cell lysis that occurs during growth in the fermentor. Consequently, production of many proteins, particularly heterologously expressed proteins, depends on the amount of proteolytic activity they are exposed to during expression and puri- fication, and the use of strains that are deficient in proteases can significantly improve overall yields. The proteolytic activities of the yeast S. cerevisiae have been characterized in detail (3), and they appear to be similar in Pichia pastoris. Most proteolytic activities in the yeast cell are contained within membrane-bound vacuoles. Pro- teinase A, a vacuolar, aspartyl protease, is encoded by the S. cerevisiae PEP4 gene, which is capable of self-activation, as well as subsequent activation of additional vacuolar proteases, such as carboxypeptidase Y and proteinase B.