Cloning and expression of the full-length cDNAS encoding human liver class 1 and class 2 aldehyde dehydrogenase.
The amino acid sequences of both human class 1 and 2 aldehyde dehydrogenase (ALDH) and the sequences of the genes coding for them are known. Based on this sequence data, we designed primers and isolated the full-length cDNAs encoding both isozymes from a human liver mRNA pool. cDNAs were subcloned in the plasmid pT7-7 and expressed in Escherichia coli with a yield of approximately 3 mg ALDH protein/liter of cell culture, although only one-third of the enzyme was soluble. The soluble recombinantly expressed ALDHs were purified to homogeneity using a hydroxyacetophenone-Sepharose affinity column. The mitochondrial isozyme had a subunit molecular weight of 55 kDa, an isoelectric point of 4.9, and a specific activity of 1.10 units/mg, which were in good agreement with that from the native enzyme. The expressed cytosolic isozyme had the same subunit molecular weight (55 kDa) and pI (5.4) as that reported for the native enzyme and had a specific activity of 0.26 units/mg. The expressed mitochondrial isozyme could be recognized by antibodies raised against rat mitochondrial ALDH, whereas the cytosolic isozyme could be recognized by antibody raised against horse cytosolic ALDH.
Zheng
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CF
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153.
Wang
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TT
Weiner
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H
D000444:Aldehyde Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology; D018076:DNA, Complementary / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation / Q000502:physiology; D006801:Humans; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification; D008099:Liver / Q000201:enzymology*; D008930:Mitochondria, Liver / Q000201:enzymology; D008970:Molecular Weight
D018076:DNA, Complementary; D007527:Isoenzymes; D000444:Aldehyde Dehydrogenase