Expression of interferon-gamma from hybrid yeast GPD promoters containing upstream regulatory sequences from the GAL1-GAL10 intergenic region.
The expression of human immune interferon (IFN-gamma) is toxic to yeast, resulting in low plasmid stability and copy number. The Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase gene (GPD) promoter [Bitter and Egan, Gene 32 (1984) 263-274] has been modified by introduction of upstream regulatory sequences from the yeast GAL1-GAL10 intergenic region [UASG; Guarente et al., Proc. Natl. Acad. Sci. USA 79 (1982) 7410-7414] and utilized to express IFN-gamma. In contrast to the native GPD promoter, the GPD(G) hybrid promoters are regulated by the carbon source. With glucose as the carbon source, a level of expression is observed which is much lower than that obtained with the native GPD promoter. Expression of the hybrid promoters is induced approx. 150- to 200-fold in shaker flask cultures by growth in galactose and similar levels of expression are observed after growth in lactate plus galactose. However, full galactose induction is not observed in the presence of glucose.? Utilization of these regulated promoters has allowed maintenance of plasmids at high copy number with glucose as the carbon source and, after induction with galactose, production of IFN-gamma mRNA at levels more than ten times higher than the native yeast PGK gene transcript. In contrast, the native GPD promoter directs comparable levels of expression when grown in either glucose or galactose resulting in low plasmid copy number and a correspondingly lower IFN-gamma transcript abundance. It is demonstrated that nucleotide sequences more than 240 bp upstream from the TATA box are required for optimal activity of the native GPD promoter.(ABSTRACT TRUNCATED AT 250 WORDS)
Bitter
G A
GA
Amgen Inc., Thousand Oaks, CA 91320.
Egan
K M
KM
D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D005690:Galactose / Q000096:biosynthesis*; D005796:Genes; D005800:Genes, Fungal; D005822:Genetic Vectors; D005954:Glucosephosphate Dehydrogenase / Q000235:genetics*; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development
D007371:Interferon-gamma; D005954:Glucosephosphate Dehydrogenase; D004262:DNA Restriction Enzymes; D005690:Galactose