Characterization of recombinant human protein C inhibitor expressed in Escherichia coli

Sophie M. Réhault, Margareta Zechmeister-Machhart, Yolanda M. Fortenberry, Julia Malleier, Nikki M. Binz, Scott T. Cooper, Margarethe Geiger, Frank C. Church

Research output: Contribution to journalArticle

Abstract

The serine protease inhibitor (serpin) protein C inhibitor (PCI; also named plasminogen activator inhibitor-3) regulates serine proteases in hemostasis, fibrinolysis, and reproduction. The biochemical activity of PCI is not fully defined partly due to the lack of a convenient expression system for active rPCI. Using pET-15b plasmid, Ni2+-chelate and heparin-Sepharose affinity chromatography steps, we describe here the expression, purification and characterization of wild-type recombinant (wt-rPCI) and two inactive mutants, R354A (P1 residue) and T341R (P14 residue), expressed in Escherichia coli. Wild-type rPCI, but not the two mutants, formed a stable bimolecular complex with thrombin, activated protein C and urokinase. In the absence of heparin, wt-rPCI-thrombin, -activated protein C, and -urokinase inhibition rates were 56.7, 3.4, and 2.3×104 M-1 min-1, respectively, and the inhibition rates were accelerated 25-, 71-, and 265-fold in the presence of 10 μg/mL heparin for each respective inhibition reaction. The stoichiometry of inhibition (SI) for wt-rPCI-thrombin was 2.0, which is comparable to plasma-derived PCI. The present report describes for the first time the expression and characterization of recombinant PCI in a bacterial expression system and demonstrates the feasibility of using this system to obtain adequate amounts of biologically active rPCI for future structure-function studies.

Original languageEnglish (US)
Pages (from-to)57-65
Number of pages9
JournalBiochimica et Biophysica Acta - Proteins and Proteomics
Volume1748
Issue number1
DOIs
StatePublished - Apr 15 2005
Externally publishedYes

Keywords

  • Heparin
  • Purification
  • Recombinant protein C inhibitor
  • Serpin
  • Thrombin

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biophysics
  • Biochemistry
  • Molecular Biology

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  • Cite this

    Réhault, S. M., Zechmeister-Machhart, M., Fortenberry, Y. M., Malleier, J., Binz, N. M., Cooper, S. T., Geiger, M., & Church, F. C. (2005). Characterization of recombinant human protein C inhibitor expressed in Escherichia coli. Biochimica et Biophysica Acta - Proteins and Proteomics, 1748(1), 57-65. https://doi.org/10.1016/j.bbapap.2004.12.003