A URA3-promoter deletion in a pYES vector increases the expression level of a fungal lipase in Saccharomyces cerevisiae.
A simple deletion of the URA3 promoter from a Saccharomyces cerevisiae expression plasmid was performed. The promoter-deleted plasmid is shown to have an increased expression level of a fungal lipase gene. The deletion probably causes a poor expression of the URA3 selection marker, probably resulting in a higher copy number per cell of the plasmid. This higher copy number can increase the transcript level per cell and there by the expression level. In the case of the fungal lipase gene, the expression level with defined inoculum is increased at least three times. The principle is most likely similar to the LEU2d plasmids described previously. A part of the 2-micron origin of the pYES type plasmid was also deleted by the URA3 promoter deletion without affecting transformation frequency. The URA3 promoter can easily be deleted from most pYES type plasmids by the described method.
Okkels
J S
JS
Novo Nordisk Enzyme Screening Symbion, København, Denmark. JSO@novo.dk
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D008049:Lipase / Q000096:biosynthesis* / Q000235:genetics; D003904:Mitosporic Fungi / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis*; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae; D017384:Sequence Deletion
D005656:Fungal Proteins; D011994:Recombinant Proteins; D008049:Lipase