2-MU-M VECTORS CONTAINING THE SACCHAROMYCES-CEREVISIAE METALLOTHIONEIN GENE AS A SELECTABLE MARKER - EXCELLENT STABILITY IN COMPLEX MEDIA, AND HIGH-LEVEL EXPRESSION OF A RECOMBINANT PROTEIN FROM A CUP1-PROMOTER-CONTROLLED EXPRESSION CASSETTE IN CIS

We have constructed 2-mu m-based yeast expression vectors containing a copy of the metallothionein (CUP1) gene of Saccharomyces cerevisine a s a semi-dominant, selectable marker. When used for the expression of the thrombin inhibitor hirudin, originally derived from the leech Hiru do medicinalis, these vectors displayed the following characteristics. (1) In the presence of copper salts, they were mitotically more stabl e than similarly designed control vectors lacking the CUP1 gene. In co pper-sensitive host strains, the apparent plasmid stability was 100%, even in complex media and during fed-batch fermentation for an extende d period of time. (2) Use of the CUP1-stabilized plasmids improved the production of hirudin by both copper-sensitive and copper-resistant h osts. The highest hirudin titers were obtained with a Delta CUP1 host. (3) Copper selection resulted in a moderate increase in average plasm id copy numbers (up to two-fold) as assessed by measuring hirudin expr ession from a constitutive promoter (GAPFL). This effect was most noti ceable if the vector showed an asymmetric segregation pattern (i.e., h igh rates of plasmid loss in the absence of copper). (4) The CUP1 mark er proved particularly useful in combination with a CUP1-promoter-cont rolled expression cassette on the same plasmid. In such a set-up. the rates of transcription of the heterologous protein and that of the sel ectable marker are tightly linked. Therefore, an increase in selective pressure directly provokes an increase in product yields. In a copper -sensitive host strain, this plasmid design allowed for the production of very high amounts of biologically active hirudin. Our results clea rly establish the utility of the CUP1 marker in the construction of st able yeast expression vectors.