Purification and characterization of active and latent forms of recombinant plasminogen activator inhibitor 1 produced in Escherichia coli.
Plasminogen activator inhibitor 1 (PAI-1), the principal physiological inhibitor of tissue plasminogen activator (tPA), is a protein of 379 amino acids and belongs to the SERPIN family of serine protease inhibitors. We have previously described methods to express [Sisk et al. (1990) Gene 96, 305-309] and purify [Reilly et al. (1990) J. Biol. Chem. 265, 9570-9574] a highly active form of the protein in substantial amounts, from Escherichia coli. Further analyses of this material showed the presence of small but significant amounts of latent rPAI-1. The present paper describes for the first time purification of latent and active forms of rPAI-1 from a single preparation, as well as the functional and structural characteristics of the two forms. Latent rPAI-1, which has properties similar to the latent forms described by other groups, was separated from active rPAI-1 by high-resolution ion-exchange chromatography or by affinity chromatography using immobilized anhydrotrypsin. It had low intrinsic activity (< 5% of active rPAI-1) and was partially reactivated by guanidine hydrochloride treatment or by incubation with vitronectin. Conversion of the active rPAI-1 to the latent form was influenced by temperature and additives including sucrose, EDTA, and arginine. Active and latent rPAI-1 did not show any obvious differences in their primary structures and displayed remarkably similar secondary structures as determined by circular dichroism spectral analyses. However, they did exhibit differences in tryptophan fluorescence, suggesting tertiary structural differences between the two forms.
Seetharam
R
R
Dupont Merck Pharmaceutical Company, Experimental Station, Wilmington, Delaware 19880-0400.
Dwivedi
A M
AM
Duke
J L
JL
Hayman
A C
AC
Walton
H L
HL
Huckins
N R
NR
Kamerkar
S M
SM
Corman
J I
JI
Woodeshick
R W
RW
Wilk
R R
RR
D000595:Amino Acid Sequence; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004355:Drug Stability; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism; D015870:Gene Expression; D006023:Glycoproteins / Q000494:pharmacology; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification*; D013050:Spectrometry, Fluorescence; D014357:Trypsin / Q000378:metabolism; D019096:Vitronectin
D006023:Glycoproteins; D006146:Guanidines; D010446:Peptide Fragments; D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins; D019096:Vitronectin; D014357:Trypsin; D019791:Guanidine