[33] Expression and secretion vectors for yeast

The chapter focuses on methods used to efficiently express and secrete biologically active proteins from Saccharomyces cereoisiae. The chapter describes yeast expression vectors utilizing episomal vectors. The expression cassettes from these vectors can also be integrated into the yeast chromosome, where they are presented at controlled copy number, exhibiting a high degree of mitotic stability. The chapter discusses extrachromosomal replication vectors, centromere plasmids, and the assembly of expression cassettes. One of the advantages of a secretion system is the production of heterologous proteins with authentic NH2 termini. The methods utilized to analyze heterologous protein secretion are the same as those used for proteins expressed directly in the cytoplasm (direct expression vectors) The chapter describes several related procedures including the Mung Bean nuclease digestion, the S1 nuclease digestion of cohesive termini, and the analysis of carbohydrate additions to heterologous proteins.