Purification of active human plasminogen activator inhibitor 1 from Escherichia coli

Abstract

Plasminogen activator inhibitor 1 (PAI-1) inhibits both tissue-type plasminogen activator (tPA) and urokinasetype 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 Escherichiu 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 :I complexes with uPA and also with the single-and two-chain forms of tPA. Kinetic analysis demonstrated that the procaryoteproduced 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 lo7 M-' s-' . In contrast to PAL-1 from eucaryotic cells, the PAI-1 from E. eoli 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°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 PAT-1 from eucaryotic cells, suggesting that it bound to the PAI-1-binding protein (vitronectin).The generation of controlled proteolytic activity is an important feature of many physiological processes [l]. The plasminogen activator (PA) system is a general proteolytic system that has been shown to be involved in such diverse processes as fibrinolysis, ovulation, cellular migration, angiogenesis and tumor metastasis [2 -61. There are two forms of plasminogen activators in mammals, tissue-type PA (tPA) and urokinase-type PA (uPA). Both PAS convert the inactive zymogen, plasminogen, into the broad-specificity protease plasmin [2]. Regulation of the PA system occurs primarily at the level of PA synthesis and activity [3]. Factors, such as hormones and growth factors have been shown to regulate the PA system by directly modulating gene expression 12, 4, 61. although there are also factors that both potentiate and inhibit the activity of the PA system following release of PAS from cells [2, 31. There are at ...

Plasminogen‐activator inhibitor type 2 (PAI‐2) is a spontaneously polymerising SERPIN

“…After chromatography, the total inhibitory activity and amount of antigen in each fraction was measured. As previously shown [33], recombinant PAI-1 elutes in two peaks, one peak with an apparent molecular mass of less than 60 kDa, representing free PAI-1, and one peak with an apparent molecular mass greater than 100 kDa, representing PAI-1 bound to vitronectin (Fig. 4A).…”

“…Using PAI-1 as a positive control, we therefore investigated whether purified recombinant PAI-2 could bind to vitronectin-coated microtiter plates [33]. While active PAI-1 was found to bind to vitronectin, we could not detect any binding of the low-molecular-mass or high-molecular-mass forms of PAI-2 (data not shown).…”

“…We therefore investigated whether the purified recombinant lowmolecular-mass and high-molecular-mass forms of PAI-2 could bind to purified vitronectin in vitro. We used conditions under which PAI-1 binds to vitronectin and analysed for binding to vitronectin using both vitronectin-coated microtiter plates and a gel-filtration assay [33]. Although binding of PAI-1 to vitronectin was detected with both assays, none of the PAI-2 forms were found to bind to vitronectin.…”

“…All the constructs were produced by the shotgun-ligation method [32] as described [33]. The accuracy of the constructs were determined by DNA sequence analysis.…”

“…The accuracy of the constructs were determined by DNA sequence analysis. The eucaryotic expression vector containing the PAI-1 signal sequence in-frame fused to PAT-2 cDNA (pPM4) was constructed as described [33].…”