Expression of human plasminogen activator inhibitor type-1 (PAI-1) in Escherichia coli as a soluble protein comprised of active and latent forms. Isolation and crystallization of latent PAI-1.
Expression of human recombinant plasminogen activator inhibitor type-1 (PAI-1) in Escherichia coli has led to crystallization of 'latent' PAI-1. Cleavage with restriction endonucleases of a cDNA clone encoding PAI-1 yielded an 1127 base pair fragment encoding residues 2-376 of the 379 amino acid serpin. Synthetic DNA linkers were ligated to the 5' and 3' ends of the subclone to add an initiation codon and restore the full coding sequence, and the resulting semisynthetic gene was incorporated into an expression plasmid, pPAIST-7, under the control of the E. coli trp promoter. Transformation of E. coli GE81 with pPAIST-7 led to expression of unglycosylated PAI-1. Lysates of expression cultures contained PAI-1 activity and PAI-1 protein with the predicted Mr. Unglycosylated PAI-1 from E. coli exhibited characteristic properties of authentic PAI-1: (1) it was recovered in both active and inactive (latent) forms; (2) its activity declined during incubation at 37 degrees C; (3) latent PAI-1 was activated by treatment with 4 M guanidine hydrochloride; (4) reactivated PAI-1 formed a detergent-stable complex with tissue plasminogen activator. Latent PAI-1 accounted for more than 85% of PAI-1 in cell lysates and was purified by ammonium sulfate fractionation, anion-exchange chromatography and hydrophobic interaction chromatography. The purified latent PAI-1 was crystallized.
Franke
A E
AE
Pfizer Central Research, Groton, CT 06340.
Danley
D E
DE
Kaczmarek
F S
FS
Hawrylik
S J
SJ
Gerard
R D
RD
Lee
S E
SE
Geoghegan
K F
KF
D000596:Amino Acids / Q000032:analysis; D002852:Chromatography, Ion Exchange; D003461:Crystallography; D004926:Escherichia coli; D015870:Gene Expression; D006801:Humans; D010957:Plasmids; D015849:Plasminogen Inactivators / Q000302:isolation & purification*; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship
D000596:Amino Acids; D015849:Plasminogen Inactivators; D011994:Recombinant Proteins