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

Arthur E. Franke, Dennis E. Danley, Frank S. Kaczmarek, Steven J. Hawrylik, Robert D. Gerard, S. Edward Lee, Kieran F. Geoghegan

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

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°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.

Original languageEnglish (US)
Pages (from-to)16-23
Number of pages8
JournalBiochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
Volume1037
Issue number1
DOIs
StatePublished - Jan 19 1990

Fingerprint

Plasminogen Activator Inhibitor 1
Crystallization
Escherichia coli
Proteins
Chromatography
Replication Protein C
Serpins
Initiator Codon
DNA Restriction Enzymes
Guanidine
Ammonium Sulfate
Tissue Plasminogen Activator
Fractionation
Hydrophobic and Hydrophilic Interactions
Base Pairing
Detergents
Anions
Ion exchange
Plasmids
Complementary DNA

Keywords

  • Enzyme crystallization
  • Latent enzyme
  • Over expression
  • Plasminogen activator inhibitor 1

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology
  • Structural Biology

Cite this

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. / Franke, Arthur E.; Danley, Dennis E.; Kaczmarek, Frank S.; Hawrylik, Steven J.; Gerard, Robert D.; Lee, S. Edward; Geoghegan, Kieran F.

In: Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular, Vol. 1037, No. 1, 19.01.1990, p. 16-23.

Research output: Contribution to journalArticle

Franke, Arthur E. ; Danley, Dennis E. ; Kaczmarek, Frank S. ; Hawrylik, Steven J. ; Gerard, Robert D. ; Lee, S. Edward ; Geoghegan, Kieran F. / 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. In: Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular. 1990 ; Vol. 1037, No. 1. pp. 16-23.
@article{dbe043da836d4299848125183d978950,
title = "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",
abstract = "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°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.",
keywords = "Enzyme crystallization, Latent enzyme, Over expression, Plasminogen activator inhibitor 1",
author = "Franke, {Arthur E.} and Danley, {Dennis E.} and Kaczmarek, {Frank S.} and Hawrylik, {Steven J.} and Gerard, {Robert D.} and Lee, {S. Edward} and Geoghegan, {Kieran F.}",
year = "1990",
month = "1",
day = "19",
doi = "10.1016/0167-4838(90)90096-X",
language = "English (US)",
volume = "1037",
pages = "16--23",
journal = "Biochimica et Biophysica Acta - Proteins and Proteomics",
issn = "1570-9639",
publisher = "Elsevier",
number = "1",

}

TY - JOUR

T1 - 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

AU - Franke, Arthur E.

AU - Danley, Dennis E.

AU - Kaczmarek, Frank S.

AU - Hawrylik, Steven J.

AU - Gerard, Robert D.

AU - Lee, S. Edward

AU - Geoghegan, Kieran F.

PY - 1990/1/19

Y1 - 1990/1/19

N2 - 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°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.

AB - 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°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.

KW - Enzyme crystallization

KW - Latent enzyme

KW - Over expression

KW - Plasminogen activator inhibitor 1

UR - http://www.scopus.com/inward/record.url?scp=0025124588&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0025124588&partnerID=8YFLogxK

U2 - 10.1016/0167-4838(90)90096-X

DO - 10.1016/0167-4838(90)90096-X

M3 - Article

C2 - 2403813

AN - SCOPUS:0025124588

VL - 1037

SP - 16

EP - 23

JO - Biochimica et Biophysica Acta - Proteins and Proteomics

JF - Biochimica et Biophysica Acta - Proteins and Proteomics

SN - 1570-9639

IS - 1

ER -