Open AccessPurification of active human plasminogen activator inhibitor 1 from Escherichia coli
Author(s) -
LAWRENCE Daniel,
STRANDBERG Leif,
GRUNDSTRÖM Thomas,
NY Tor
Publication year - 1989
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1989.tb15238.x
Subject(s) - chinese hamster ovary cell , plasminogen activator , recombinant dna , microbiology and biotechnology , t plasminogen activator , escherichia coli , expression vector , plasminogen activator inhibitor 1 , biology , transfection , complementary dna , activator (genetics) , tissue plasminogen activator , cell culture , biochemistry , chemistry , gene , receptor , endocrinology , genetics
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 procarvotic expression systems for PAI‐1 and characterized the recombinant glycosylated and non‐glycosylated products, together with a non‐recombinant natural control, produced in the histösarcoma 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 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 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°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).