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. 1990 Nov 1;271(3):755–760. doi: 10.1042/bj2710755

cDNA cloning and expression in Xenopus laevis oocytes of pig renal dipeptidase, a glycosyl-phosphatidylinositol-anchored ectoenzyme.

E Rached 1, N M Hooper 1, P James 1, G Semenza 1, A J Turner 1, N Mantei 1
PMCID: PMC1149627  PMID: 2173907

Abstract

Clones expressing renal dipeptidase (EC 3.4.13.11) have been isolated from a pig kidney cortex cDNA library after employing the polymerase chain reaction technique to amplify a region of the dipeptidase cDNA. The complete primary sequence of the enzyme has been deduced from a full length cDNA clone. This predicts a protein of 409 amino acids, a cleavable N-terminal signal sequence of 16 residues and two N-linked glycosylation sites. At the C-terminus of the predicted sequence is a stretch of mainly hydrophobic amino acids which is presumed to direct the attachment of the glycosyl-phosphatidylinositol membrane anchor. Expression of the mRNA for pig renal dipeptidase in Xenopus laevis oocytes led to the production of a disulphide-linked dimeric protein of subunit Mr 48,600 which was recognized by a polyclonal antiserum raised to renal dipeptidase purified from pig kidney cortex. Bacterial phosphatidylinositol-specific phospholipase C released renal dipeptidase from the surface of the oocytes and converted the amphipathic detergent-solubilized form of the dipeptidase to a hydrophilic form, indicating that Xenopus laevis oocytes can process expressed proteins to their glycosyl-phosphatidylinositol anchored form.

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