Ludwig D L, Ugolini S, Bruschi C V
Microbiology Department, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy.
Gene. 1993 Sep 30;132(1):33-40. doi: 10.1016/0378-1119(93)90511-z.
The best candidate for a high-copy-number and mitotic stability expression system in yeast is the endogenous 2 microns plasmid. Nevertheless, derivatives of the 2 microns 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 expression system containing a 4.5-kb inducible expression cassette inserted into the 2 microns plasmid and selected in cells utilizing a carrier plasmid which is subsequently lost via FRT/Flp recombination. The non-selectable 2 micron plasmid, containing the cassette, was found to be stably maintained in cells, without selection, at high copy number. The dynamics 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 (beta Gal) is driven by the hybrid, galactose-inducible promoter GAL10::pMF alpha 1. Upon induction, beta Gal was secreted into the periplasm and culture supernatant at levels which could be detected directly from Coomassie blue-stained SDS-PAGE. Furthermore, plasmid-containing cells could be maintained directly on rich YPD medium and identified either by utilizing XGal or by observing inhibition of colony growth on YPGal solid medium. The cassette was designed for direct, high-level, inducible expression of cloned genes downstream from the MF alpha 1 signal sequence, with or without a C-terminal lacZ fusion. This vector represents the first demonstration of a non-selectable, mitotically stable, episomal plasmid system capable of expressing recombinant proteins at high levels.(ABSTRACT TRUNCATED AT 250 WORDS)
