Fluorescence based assay of GAL system in yeast Saccharomyces cerevisiae.
The GAL1 promoter is one of the strongest inducible promoters in the yeast Saccharomyces cerevisiae. In order to improve recombinant protein production we have developed a fluorescence based method for screening and evaluating the contribution of various gene deletions to protein expression from the GAL1 promoter. The level of protein synthesis was determined in 28 selected mutant strains simultaneously, by direct measurement of fluorescence in living cells using a microplate reader. The highest, 2.4-fold increase in GFP production was observed in a gal1 mutant strain. Deletion of GAL80 caused a 1.3-fold increase in fluorescence relative to the isogenic strain. GAL3, GAL4 and MTH1 gene deletion completely abrogated GFP synthesis. Growth of gal7, gal10 and gal3 also exhibited reduced fitness in galactose medium. Other genetic perturbations affected the GFP expression level only moderately. The fluorescence based method proved to be useful for screening genes involved in GAL1 promoter regulation and provides insight into more efficient manipulation of the GAL system.
Stagoj
Mateja Novak
MN
National Institute of Chemistry, Laboratory for Biosynthesis and Biotransformation, Hajdrihova 19, SI-1000 Ljubljana, Slovenia. mateja.novak@ki.si
Comino
Aleksandra
A
Komel
Radovan
R
D005690:Galactose / Q000235:genetics* / Q000378:metabolism; D017353:Gene Deletion; D015870:Gene Expression; D005800:Genes, Fungal; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D009154:Mutation; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis / Q000235:genetics; D013050:Spectrometry, Fluorescence
D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D005690:Galactose