SEPARATION OF ACTIVE AND INACTIVE FORMS OF RECOMBINANT HUMAN PLASMINOGEN-ACTIVATOR INHIBITOR TYPE-1 (PAI-1) EXPRESSED IN CHINESE-HAMSTER OVARY CELLS - COMPARISON WITH NATIVE HUMAN PAI-1

Human plasminogen activator inhibitor type 1, PAI-1, was expressed in Chinese hamster ovary cells. A production level of 10-15 mg latent PAI -1 per liter of media was achieved after methotrexate amplification. L atent recombinant PAI-1 was purified by two chromatographic steps, cat ion exchange chromatography on CM-Sepharose and affinity chromatograph y on heparin-Sepharose. The obtained latent PAI-1 was approximately 90 -95% pure showing one homogenous peak upon size-exclusion chromatograp hy. However, four different isoforms due to different degrees of sialy lation could be seen upon isoelectric focusing. Purified latent PAI-1 was activated by incubation in 6 M guanidine-HCl. By this method, 40-6 0% of PAI-1 was converted to an active form after removing the denatur ant. The active fraction of PAI-1 was separated from inactive material by size exclusion chromatography on Superdex 200. Active PAI-1 migrat ed as expected for a 43-kDa large protein, while inactive PAI-1 migrat ed as larger protein complexes, suggesting that the remaining inactive PAI-1 was in the form of aggregates. This method for the separation o f active and inactive PAI-1 could also be used for activated native PA I-1 prepared from human endothelial cells. Active recombinant PAI-1 wa s remarkably stable at pH 5.5, both when stored on ice and when stored at room temperature. (C) 1994 Academic Press, Inc.