Purification and Characterization of Active and Stable Recombinant Plasminogen‐Activator Inhibitor Accumulated at High Levels in Escherichia coli

Abstract

Plasminogen‐activator inhibitor type 1 (PAI‐l), the primary physiological inhibitor of tissue‐type plasminogen activator, is an unusual member of the serine protease inhibitor (serpin) superfamily in that it spontaneously converts to a latent form lacking activity. This latent form can be reactivated by denaturation and refolding, but the activation is usually incomplete and often leads to aggregation of the protein. In this study we have developed a high‐level expression system that leads to the accumulation of PAI‐1 at 30–50% total microbial protein. We have developed a single‐step purification protocol which can be completed in a few hours, yielding approximately 20 mg purified recombinant PAI‐Mitre culture. The purified PAI‐1 was 80–100% active and was stable upon incubation at 37°C with a half‐life of approximately 48 h. At 20°C, PAI‐1 activity was stable for a week and at 4°C it retained its activity completely for up to two months. Freezing caused significant loss of activity. The stability of PAI‐1 activity was found to be dependent on pH and ionic strength, being most stable at pH 5.6 and at an ionic strength of 1 M salt. We show that by a combination of high‐level expression and rapid purification under optimum conditions, it is possible to produce active and stable PAI‐1 in high yield. Copyright © 1994, Wiley Blackwell. All rights reserved