PURIFICATION AND CHARACTERIZATION OF RECOMBINANT RABBIT PLASMINOGEN-ACTIVATOR INHIBITOR-1 EXPRESSED IN SACCHAROMYCES-CEREVISIAE

The rabbit plasminogen activator inhibitor-1 (PAI-1) cDNA has been isolated from a rabbit corneal cell cDNA library. The cDNA encodes a 402-amino acid (AA) protein that shares an overall 66% AA sequence identity with the rat, mouse, bovine and human forms of PAI-1 and exhibits the greatest AA sequence identity (85 %) with human PAI-1. Three potential N-linked glycosylation sites and the P1, P1' reactive centre of PAI-1 are conserved among all five species of animals. The cDNA encoding the proposed mature form of rabbit PAI-1 was expressed in Saccharomyces cerevisiae as an intracellular, non-glycosylated protein. The purified, recombinant rabbit PAI-1 (R-rPAI-1) has an apparent M(r) of 39 100 and exists primarily in a latent form which can be activated by guanidine HCl treatment. Activated R-rPAI-1 exhibits in vitro functional properties which are virtually indistinguishable from a recombinant, non-glycosylated form of human PAI-1 and from fully glycosylated, native human PAI-1.