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. Latent recombinant PAI-1 was purified by two chromatographic steps, cation exchange chromatography on CM-Sepharose and affinity chromatography on heparin-Sepharose. The obtained latent PAI-1 was approximately 90-95% pure showing one homogenous peak upon size-exclusion chromatography. However, four different isoforms due to different degrees of sialylation could be seen upon isoelectric focusing. Purified latent PAI-1 was activated by incubation in 6 M guanidine-HCl. By this method, 40-60% of PAI-1 was converted to an active form after removing the denaturant. The active fraction of PAI-1 was separated from inactive material by size exclusion chromatography on Superdex 200. Active PAI-1 migrated as expected for a 43-kDa large protein, while inactive PAI-1 migrated as larger protein complexes, suggesting that the remaining inactive PAI-1 was in the form of aggregates. This method for the separation of active and inactive PAI-1 could also be used for activated native PAI-1 prepared from human endothelial cells. Active recombinant PAI-1 was remarkably stable at pH 5.5, both when stored on ice and when stored at room temperature.
Strömqvist
M
M
Symbicom AB, Umeå, Sweden.
Andersson
J O
JO
Boström
S
S
Deinum
J
J
Ehnebom
J
J
Enquist
K
K
Johansson
T
T
Hansson
L
L
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002241:Carbohydrates / Q000032:analysis; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D004789:Enzyme Activation; D004795:Enzyme Stability; D006801:Humans; D007525:Isoelectric Focusing; D009439:Neuraminidase / Q000378:metabolism; D017395:Plasminogen Activator Inhibitor 1 / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification
D000596:Amino Acids; D002241:Carbohydrates; D018076:DNA, Complementary; D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins; D009439:Neuraminidase