Purification and characterization of active and stable recombinant plasminogen-activator inhibitor accumulated at high levels in Escherichia coli.
Plasminogen-activator inhibitor type 1 (PAI-1), 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-1/litre culture. The purified PAI-1 was 80-100% active and was stable upon incubation at 37 degrees C with a half-life of approximately 48 h. At 20 degrees C, PAI-1 activity was stable for a week and at 4 degrees 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.
Sancho
E
E
Department of Molecular and Cell Biology, University of Aberdeen, Scotland.
Tonge
D W
DW
Hockney
R C
RC
Booth
N A
NA
D001120:Arginine / Q000494:pharmacology; D001483:Base Sequence; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017395:Plasminogen Activator Inhibitor 1 / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D012965:Sodium Chloride / Q000494:pharmacology; D013696:Temperature
D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins; D012965:Sodium Chloride; D001120:Arginine