Heterologous expression of human 5-aminolevulinate dehydratase in Saccharomyces cerevisiae.
A cDNA coding for human 5-amino-levulinate dehydratase was placed in a yeast expression vector under the control of the GAL10 promoter. The resulting multicopy plasmid was then used to transform a yeast mutant which contains a defective hem2 gene coding for 5-aminolevulinate dehydratase. Expression of the human cDNA was shown in four ways: (1) restoration of normal growth on glycerol/galactose as primary carbon source, (2) decrease in intracellular 5-aminolevulinic acid concentration, (3) restoration of cytochrome biosynthesis and (4) direct, in situ assay of 5-aminolevulinic acid dehydratase. Curing transformed cells of plasmid restored the hem2 mutant phenotype. This heterologous system could be used to produce large quantities of human 5-aminolevulinic acid dehydratase for physical and biochemical studies.
Schauer
W E
WE
Biotechnology Center, University of Colorado, Colorado Springs 80933-7150.
Mattoon
J R
JR
D000483:Alleles; D003580:Cytochromes / Q000096:biosynthesis; D004247:DNA / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D005796:Genes; D006801:Humans; D009154:Mutation; D010957:Plasmids; D000623:Porphobilinogen Synthase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D014170:Transformation, Genetic
D003580:Cytochromes; D004247:DNA; D000623:Porphobilinogen Synthase