Purification of recombinant plasminogen activator inhibitor-1 in the active conformation by refolding from inclusion bodies.
Plasminogen activator inhibitor-1 (PAI-1) acts as the major inhibitor of fibrinolysis by inhibiting tissue-type and urokinase-type plasminogen activators. Although it shares a common tertiary structure with other serine protease inhibitors, PAI-1 is unique in its conformational lability, which allows conversion of the active form to the latent conformation under physiological conditions. Therefore, recombinant PAI-1 expressed in eukaryotic or prokaryotic cells almost always contains its inactive, latent form, with very low specific activity. In this study, we developed a simple and efficient method for purifying the active form of recombinant PAI-1 rather than the latent conformation from PAI-1 overexpressing Escherichia coli cells. The overall level of expression and the amount of PAI-1 found in inclusion bodies were found to increase with culture temperature and with time after induction. Refolding of unfolded PAI-1 from inclusion bodies and ion-exchange column chromatography were sufficient to purify PAI-1. The purified protein yielded a single, 43kDa protein band upon SDS-polyacrylamide gel electrophoresis, and it efficiently inhibited tissue-type and urokinase-type plasminogen activators similar to PAI-1 from natural sources. Activity measurements showed that PAI-1 purified from inclusion bodies exhibited a specific activity near the theoretical maximum, unlike PAI-1 prepared from cytosolic fractions. Conformational analysis by urea gel electrophoresis also indicated that the PAI-1 protein purified from inclusion bodies was indeed in its active conformation.
Lee
Hak-Joo
HJ
Department of Molecular Biology, Sejong University, 98 Gunja-dong, Kwangjin-gu, 143-747, Seoul, South Korea.
Im
Hana
H
D002458:Cell Fractionation; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D015870:Gene Expression / Q000187:drug effects; D005822:Genetic Vectors / Q000235:genetics; D019791:Guanidine / Q000737:chemistry; D006639:Histidine; D006801:Humans; D015151:Immunoblotting; D002479:Inclusion Bodies / Q000737:chemistry; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D010920:Placenta / Q000737:chemistry; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D010960:Plasminogen Activators / Q000378:metabolism; D011247:Pregnancy; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D013053:Spectrophotometry; D013696:Temperature
D018076:DNA, Complementary; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D006639:Histidine; C049024:H-hexahydrotyrosyl-alanyl-arginine-4-nitroanilide; D010960:Plasminogen Activators; D019791:Guanidine