Isolation of a cDNA encoding functional Drosophila alcohol dehydrogenase in Escherichia coli and purification of the bacterially produced enzyme.
Because of the severe limitations on growing large quantities of Drosophila affinidisjuncta in the laboratory, direct purification of alcohol dehydrogenase (ADH) from this species has proven impossible. As an alternative source of this enzyme, a cDNA encoding functional ADH was isolated from a newly constructed cDNA library made from larval poly(A)-containing RNA. The cDNA was recovered by virtue of its hybridization to a previously isolated genomic ADH gene. Nucleotide sequence analysis confirmed the identity of the newly isolated cDNA. When the cDNA was inserted in the proper orientation downstream of the lac promoter on the vector pUC8, the cDNA directed the synthesis of functional ADH by the bacterial host. The bacterially produced enzyme was purified to homogeneity and used to elicit polyclonal antibodies in rabbits. The purified ADH has identical apparent subunit molecular weight to that of authentic ADH in larval fly extracts as determined by immunoblotting. Further, comparisons of the kinetic parameters of the bacterially produced enzyme and ADH activity in larval fly extracts indicate similar substrate preferences, pH dependencies, and Km values for 2-propanol and NAD. These results show that expression of a cDNA in Escherichia coli is a valid strategy for isolation of an ADH that would otherwise be difficult or impossible to purify.
Green
M M
MM
Department of Biology, University of Alabama, Tuscaloosa 35487-0344.
Fang
X M
XM
Churchill
P
P
Brennan
M D
MD
D000426:Alcohol Dehydrogenase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification*; D004330:Drosophila / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids; D011817:Rabbits; D013379:Substrate Specificity
D004247:DNA; D000426:Alcohol Dehydrogenase