cDNA cloning and expression in <i>E</i>. <i>coli</i> of a plasminogen activator inhibitor (PAI) related to a PAI produced by Hep G2 hepatoma cell

Abstract

The human hepatoma line Hep G2 produces an acid- and SDS-sensitive plasminogen activator inhibitor (PAI). This protein has been previously purified and used to raise polyclonal antibodies. This antiserum has been used to isolate cDNA clones from a human placental lambda gt11 cDNA library. The immunologically positive clones were screened for expression of recombinant proteins which inhibit urokinase activity and form an inhibitor-enzyme complex with 125I-urokinase. Two positives (lambda PAI 11.1 and lambda PAI 14.1) have been obtained. The cDNA insert of the longer isolate (lambda PAI 14.1) consists of 1962 base pairs encoding the entire mature Hep G2 PAI and a 3'-noncoding region of 801 base pairs. The clone apparently lacks portions of 5'- and 3'-untranslated sequences. The translated amino acid sequence matches the sequence obtained for the mature Hep G2 PAI and consists of 379 amino acids with a molecular mass of 42 770 Da. Interestingly, this PAI clone is quite different from the placental-type PAI-2 sequence as expected, but matches the sequence of the endothelial-type PAI (PAI-1) reported to be acid-insensitive and SDS-enhancible.