phungthiquynhanh
Junior Member
Purification and characterization of active
and stable recombinant plasminogen-activator inhibitor accumulated
at high levels in Escherichia coli
PAI-1 was identified as a member of the superfamily of serine protease inhibitors known as serpins, on the basis ofits sequence [16-181. The serpins are the major class ofinhibitors regulating the fibrinolytic, coagulation and complement cascades. They act as pseudosubstrates and form an SDS-stable covalent 1 : 1 complex with their target protease. Subsequent dissociation of the complex by primary amines yields an inactive inhibitor that has been cleaved at its reactive centre [19]. PAI-1 is a glycoprotein of apparent molecular mass 48 kDa; carbohydrate, which occurs at three Nlinked sites, accounts for approximately 13% of the molecular mass. PAI-1 consists of 379 amino acids, corresponding to a 43 kDa polypeptide chain, and is notable for the complete lack of cysteine residues [16-181. PAI-1 is unusual among the serpins in its tendency to lose activity and to assume a latent conformation. Latent PAI- 1 is so-called because it can be reactivated by exposure to denaturing agents [20]. The X-ray structure of latent PAI-1 has been solved [21] and it appears that the reactive centre is buried in the structure, rather than being exposed to the target protease, thus offering an explanation for its lack of activity. Denaturation and refolding of the molecule expose the reactive centre and yields an active inhibitor. PAI-1 is synthesized in its active form by endothelial cells in culture; once released into the culture medium PAI-1 loses activity with a half-life of 0.5-3 h [22, 231. The activity of PAI-1 can be stabilized by association with vitronectin in plasma [24] and in the extracellular matrix
and stable recombinant plasminogen-activator inhibitor accumulated
at high levels in Escherichia coli
PAI-1 was identified as a member of the superfamily of serine protease inhibitors known as serpins, on the basis ofits sequence [16-181. The serpins are the major class ofinhibitors regulating the fibrinolytic, coagulation and complement cascades. They act as pseudosubstrates and form an SDS-stable covalent 1 : 1 complex with their target protease. Subsequent dissociation of the complex by primary amines yields an inactive inhibitor that has been cleaved at its reactive centre [19]. PAI-1 is a glycoprotein of apparent molecular mass 48 kDa; carbohydrate, which occurs at three Nlinked sites, accounts for approximately 13% of the molecular mass. PAI-1 consists of 379 amino acids, corresponding to a 43 kDa polypeptide chain, and is notable for the complete lack of cysteine residues [16-181. PAI-1 is unusual among the serpins in its tendency to lose activity and to assume a latent conformation. Latent PAI- 1 is so-called because it can be reactivated by exposure to denaturing agents [20]. The X-ray structure of latent PAI-1 has been solved [21] and it appears that the reactive centre is buried in the structure, rather than being exposed to the target protease, thus offering an explanation for its lack of activity. Denaturation and refolding of the molecule expose the reactive centre and yields an active inhibitor. PAI-1 is synthesized in its active form by endothelial cells in culture; once released into the culture medium PAI-1 loses activity with a half-life of 0.5-3 h [22, 231. The activity of PAI-1 can be stabilized by association with vitronectin in plasma [24] and in the extracellular matrix
