HIGH-LEVEL HETEROLOGOUS GENE-EXPRESSION IN SACCHAROMYCES-CEREVISIAE FROM A STABLE 2-MU-M PLASMID SYSTEM

The best candidate for a high-copy-number and mitotic stability expres sion system in yeast is the endogenous 2mum plasmid. Nevertheless, der ivatives of the 2mum plasmid typically exhibit lower copy numbers and require selection for adequate maintenance within cells. We report the construction and utilization of an efficient heterologous gene expres sion system containing a 4.5-kb inducible expression cassette inserted into the 2mum plasmid and selected in cells utilizing a carrier plasm id which is subsequently lost via FRT/Flp recombination. The non-selec table 2mum plasmid, containing the cassette, was found to be stably ma intained in cells, without selection, at high copy number. The dynamic s of resolution and partitioning of this plasmid were analyzed during the course of 50 generations of growth under non-selective conditions. The heterologous lacZ reporter gene coding for beta-galactosidase (be taGal) is driven by the hybrid, galactose-inducible promoter GAL10=pMF alpha1. Upon induction, betaGal was secreted into the periplasm and cu lture supernatant at levels which could be detected directly from Coom assie blue-stained SDS-PAGE. Furthermore, plasmid-containing cells cou ld be maintained directly on rich YPD medium and identified either by utilizing XGal or by observing inhibition of colony growth on YPGal so lid medium. The cassette was designed for direct, high-level, inducibl e expression of cloned genes downstream from the MFalpha1 signal seque nce, with or without a C-terminal lacZ fusion. This vector represents the first demonstration of a non-selectable, mitotically stable, episo mal plasmid system capable of expressing recombinant proteins at high levels. By supplanting the need for synthetic medium, this system coul d provide both an efficient and cost-effective means of generating rec ombinant protein at either the laboratory or large-scale level.