Cloning and Prokaryotic Expression of a Biologically Active Human Placental Aldose Reductase

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Abstract
CDNA clones coding for human aldose reductase (AR) were isolated by antibody screening of a placental λgt11 cDNA library. The cDNA comprises the entire coding region and has a total length of 1,394 bp. The sequence deduced from the open reading frame encodes a protein of 316 amino acids and its amino acid composition is identical to the placental protein 9 (PP9), whose isolation and characterization were described by Bohn et al. (1982). The amino acid sequence of the placental human AR shows high homology to the rat AR; both proteins belong to the same protein superfamily as human liver AR, frog lens rho-crystallin, and bovine lung prostaglandin F synthase. Northern blot hybridization analysis revealed a size for the AR mRNA of approximately 1,500 bases. In addition to the full-length cDNA, one λgt11 clone was isolated which carries a putative intron of 597 bp at nucleotide position 754, corresponding to amino acid position 247. Expression of the AR cDNA in Escherichia coli resulted in the synthesis of a protein with a molecular weight of approximately 35 kD which can be immunoprecipitated specifically with antiserum raised against PP9. Despite the absence of a typical signal sequence, the human aldose reductase is partially translocated into the periplasm of the E. coli cells, where it is present in an enzymatically active form.