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 dehydrog enase (ALDH) and the sequences of the genes coding for them are known. Based on this sequence data, we designed primers and isolated the ful l-length cDNAs encoding both isozymes from a human liver mRNA pool. cD NAs were subcloned in the plasmid pT7-7 and expressed in Escherichia c oli with a yield of approximately 3 mg ALDH protein/liter of cell cult ure, although only one-third of the enzyme was soluble. The soluble re combinantly expressed ALDHs were purified to homogeneity using a hydro xyacetophenone-Sepharose affinity column. The mitochondrial isozyme ha d a subunit molecular weight of 55 kDa, an isoelectric point of 4.9, a nd 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 pl (5.4) as that report ed 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.