Yeast vector

Claim 2

A 2µm plasmid vector comprising a DNA sequence which is intended to be lost by recombination, three 2µm FLP recombination sites, of which one pair of sites is in direct orientation and the other two pairs are in indirect orientation, and a DNA sequence coding for a protein or peptide of interest, the said sequence to be lost being located between the said sites which are in direct orientation.

Claim 3

A 2µm plasmid vector according to Claim 1 which also comprises a selectable marker DNA sequence.

Claim 4

A 2µm plasmid vector according to Claim 2 comprising a complete 2µm plasmid additionally carrying (i) a bacterial plasmid DNA sequence necessary for propagation of the vector in a bacterial host; (ii) an extra 2µm FLP recombination site; (iii) a DNA sequence coding for a protein or peptide of interest; and (iv) a selectable marker DNA sequence for yeast transformation; the said bacterial plasmid DNA sequence being present and the extra FLP recombination site being created at a restriction site in one of the two inverted repeat sequences of the 2µm plasmid, the said extra FLP recombination site being in direct orientation in relation to the endogenous FLP recombination site of the said one repeat sequence, and the bacterial plasmid DNA sequence being sandwiched between the extra FLP recombination site and the endogenous FLP recombination site of the said one repeat sequence.

Claim 5

A 2µm plasmid vector according to Claim 3 wherein the restriction site is the unique Xba I site.

Claim 6

A 2µm plasmid vector according to Claim 3 or 4 wherein all bacterial plasmid DNA sequences are sandwiched as said.

Claim 7

A 2µm plasmid vector according to any of Claims 1 to 5 in which the DNA sequence coding for a protein or peptide of interest is heterologous to yeast.

Claim 8

A 2 µm plasmid vector according to Claim 6 in which the DNA sequence coding for a protein or peptide of interest is a DNA sequence coding for HSA fused at its 5ʹ terminus to a gene promoter which functions in yeast via a secretion leader sequence which functions in yeast and at its 3ʹ terminus to a transcription termination signal which functions in yeast.

Claim 9

A 2µm plasmid vector according to Claim 6 in which the DNA sequence coding for a protein or peptide of interest is the MET-HSA gene fused at its 5ʹ terminus to the GAL/CYC1 or GAL/PGK hybrid promoter, and at its 3ʹ terminus to a transcription termination signal which functions in yeast.

Claim 10

A 2µm plasmid vector according to any of Claims 1 to 5 in which the DNA sequence coding for a protein or peptide of interest is the DEX-1 gene or a DNA sequence coding for the β-glucanase of Bacillus subtilis fused at its 5ʹ terminus to a gene promoter which functions in yeast via a secretion leader sequence which functions in yeast and at its 3ʹ terminus to a transcription termination signal which functions in yeast.

Claim 11

A 2µm plasmid vector according to Claim 1 having substantially the configuration of pSAC3 as shown in Figure 3 of the accompanying drawings.

Claim 12

A process for preparing a 2µm plasmid vector according to any one of the preceding claims comprising inserting into a complete 2µm plasmid (i) a DNA sequence for selecting yeast transformants, (ii) a DNA sequence encoding a protein or peptide of interest and (iii) an insert comprising (a) bacterial plasmid DNA to allow propagation of the vector in bacteria and (b) the elements of a FLP recombination site such that an extra FLP recombination site is created in the vector and the said bacterial DNA is sandwiched between two FLP recombination sites in mutually direct orientation.

Claim 13

A process according to Claim 11 wherein the insert is inserted at the unique Xba I site of an endogenous FLP recombination site, one end of the insert carries one portion of a 2µm repeat sequence and the other end of the insert carries the remainder of the 2µm repeat sequence.

Claim 14

A 2µm plasmid vector comprising a DNA sequence encoding a protein or peptide of interest which is heterologous to yeast, the vector comprising no bacterial DNA.

Claim 15

A 2µm plasmid vector comprising a DNA sequence encoding a protein or peptide of interest which is heterologous to yeast, the vector comprising no bacterial DNA.

Claim 16

Brewing yeast or laboratory yeast transformed with a 2µm plasmid vector as claimed in any of Claims 1 to 10 and 13.

Claim 17

A protein or peptide of interest prepared by fermenting a yeast according to Claim 14.

Claim 18

A 2µm plasmid vector carrying a gene of interest inserted directly or indirectly at the Sna BI site.

Claim 19

A 2µm plasmid vector carrying a gene of interest inserted directly or indirectly at the Sna BI site.