title: Purification of active human plasminogen activator inhibitor 1 from Escherichia coli. Comparison with natural and recombinant forms purified from eucaryotic cells.
abstract: Plasminogen activator inhibitor 1 (PAI-1) inhibits both tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) and, therefore, is an important regulator of plasminogen activation. We have developed eucaryotic and procaryotic expression systems for PAI-1 and characterized the recombinant glycosylated and non-glycosylated products, together with a non-recombinant natural control, produced in the histosarcoma cell line HT 1080. For eucaryotic expression, the PAI-1 cDNA was stably transfected into chinese hamster ovary cells (CHO cells), while procaryotic expression in Escherichia coli was examined after inserting the DNA sequence encoding the mature PAI-1 protein into an inducible expression vector. Recombinant PAI-1 from CHO cells was purified approximately 50-fold in two steps and was indistinguishable from natural PAI-1. Between 3% and 4% of total cellular protein in the procaryotic expression system consisted of PAI-1, from which it was purified approximately 30-fold, with yields of between 15% and 20%. This PAI-1 formed 1:1 complexes with uPA and also with the single- and two-chain forms of tPA. Kinetic analysis demonstrated that the procaryote-produced PAI-1 had an inhibitory activity towards all three forms of PA that resembled that of natural PAI-1 with association rate constants of approximately 10(7) M-1 s-1. In contrast to PAI-1 from eucaryotic cells, the PAI-1 from E. coli had an inherent activity equal to that of guanidine/HCl-activated natural PAI-1. The activity could not be increased by treatment with denaturants suggesting that the latent form of PAI-1 was absent. However, at 37 degrees C the procaryote-produced PAI-1 lost activity at the same rate as natural PAI-1, with approximately 50% of the activity remaining after 3 h. This activity could be partially restored by treatment with 4 M guanidine/HCl. E. coli-derived PAI-1, added to human plasma and fractionated by Sephacryl S-200 chromatography, eluted in two peaks that were similar to those obtained with guanidine-activated PAI-1 from eucaryotic cells, suggesting that it bound to the PAI-1-binding protein (vitronectin).
authors.affiliation: Department of Applied Cell and Molecular Biology, University of Umeå, Sweden.
authors.forename: D
authors.initials: D
authors.lastname: Lawrence
authors.forename: L
authors.initials: L
authors.lastname: Strandberg
authors.forename: T
authors.initials: T
authors.lastname: Grundström
authors.forename: T
authors.initials: T
authors.lastname: Ny
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D005343:Fibrinolytic Agents / Q000494:pharmacology; D005796:Genes; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015345:Oligonucleotide Probes; D010957:Plasmids; D010960:Plasminogen Activators / Q000037:antagonists & inhibitors; D015849:Plasminogen Inactivators / Q000302:isolation & purification* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000302:isolation & purification; D010959:Tissue Plasminogen Activator / Q000037:antagonists & inhibitors; D014162:Transfection; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors
chemical_list: D005343:Fibrinolytic Agents; D015345:Oligonucleotide Probes; D015849:Plasminogen Inactivators; D011994:Recombinant Proteins; D010960:Plasminogen Activators; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator