New tools for high-throughput expression of fungal secretory proteins in Saccharomyces cerevisiae and Pichia pastoris.

Heterologous protein expression in yeast, mostly in Saccharomyces cerevisiae and Pichia pastoris, is a well-established and widely used technique. It typically requires the construction of an expression vector in Escherichia coli containing the foreign gene and its subsequent transformation into yeast. Although simple, this procedure has important limitations for the expression of large numbers of proteins, that is, for the generation of protein libraries. We describe here the development of a novel system for the easy and fast expression of heterologous proteins both in S. cerevisiae and in P. pastoris, under the control of the GAL1 and AOX1 promoters respectively. Expression in S. cerevisiae requires only the transformation of yeast cells with an unpurified PCR product carrying the gene to be expressed, and the expression of the same gene in P. pastoris requires only the isolation of the plasmid generated in S. cerevisiae and its transformation into this second yeast, thus making this system suitable for high-throughput projects. The system has been tested by the extracellular expression of 30 secretory fungal proteins.