AN INTEGRATING VECTOR FOR TUNABLE, HIGH COPY, STABLE INTEGRATION INTOTHE DISPERSED TY DELTA-SITES OF SACCHAROMYCES-CEREVISIAE

We have constructed a yeast integration vector targeted to chromosomal Ty delta sequences and used it to create Saccharomyces cerevisiae str ains with stable tandem integrations ranging from 1 to 30 vector copie s. The vector carries the bacterial NEO gene, allowing copy number to be tuned by varying G418 resistance, which generally increases with co py number as determined by quantitative Southern blot. Tandem integrat ion into a single site is most commonly observed, but single-copy and two-site integration is also observed. Bovine pancreatic trypsin inhib itor was constitutively expressed and secreted using the NEO-based del ta vector, and secretion levels were 2-10-fold improved relative to co mmonly used 2 mu multicopy yeast plasmids. The NEO-based Ty delta vect or is a powerful tool for stable heterologous protein expression and s ecretion in yeast.