Synthesis of Drosophila melanogaster alcohol dehydrogenase in yeast.
Expression systems for the heterologous expression of Drosophila melanogaster alcohol dehydrogenase (ADH) in Saccharomyces cerevisiae have been designed, analyzed and compared. Four different yeast/Escherichia coli shuttle vectors were constructed and used to transform four different yeast strains. Expression was detectable in ADH- yeast strains, from either a constitutive promoter, yeast ADH1 promoter (ADCp), or a regulated promoter, yeast GALp. The highest amount of D. melanogaster ADH was obtained from a multicopy plasmid with the D. melanogaster Adh gene expressed constitutively under the control of yeast ADCp promoter. The D. melanogaster enzyme was produced in cell extracts, as assessed by Coomassie blue staining and Western blotting after polyacrylamide-gel electrophoresis and it was fully active and able to complement the yeast ADH deficiency. Results show that D. melanogaster ADH subunits synthesized in yeast are able to assemble into functional dimeric forms. The synthesized D. melanogaster ADH represents up to 3.5% of the total extracted yeast protein.
Atrian
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Departament de Genètica, Universitat de Barcelona, Spain.
Gonzàlez-Duarte
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Fothergill-Gilmore
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LA
D000426:Alcohol Dehydrogenase / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D003720:Densitometry; D004331:Drosophila melanogaster / Q000201:enzymology*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005816:Genetic Complementation Test; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014170:Transformation, Genetic
D004274:DNA, Recombinant; D000426:Alcohol Dehydrogenase