Characterization of recombinant human protein C inhibitor expressed in Escherichia coli

• Published in: Biochimica et biophysica acta Vol. 1748; no. 1; pp. 57 - 65
• Main Authors: Réhault, Sophie M., Zechmeister-Machhart, Margareta, Fortenberry, Yolanda M., Malleier, Julia, Binz, Nikki M., Cooper, Scott T., Geiger, Margarethe, Church, Frank C.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 15.04.2005
• Subjects: Amino Acid Sequence; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Heparin; Heparin - metabolism; Humans; Molecular Sequence Data; Mutation; Protein C - antagonists & inhibitors; Protein C - metabolism; Protein C Inhibitor - genetics; Protein C Inhibitor - metabolism; Purification; Recombinant protein C inhibitor; Recombinant Proteins - metabolism; Sequence Alignment; Serine Proteinase Inhibitors - genetics; Serine Proteinase Inhibitors - metabolism; Serpin; Thrombin; Thrombin - antagonists & inhibitors; Thrombin - metabolism; Urokinase-Type Plasminogen Activator - antagonists & inhibitors; Urokinase-Type Plasminogen Activator - metabolism; rPCI; Serpin; Purification; APC; P14-rPCI; IPTG; Recombinant protein C inhibitor; Thrombin; LB medium; Heparin; Ni-NTA; P1-rPCI
• ISSN: 1570-9639; 0006-3002; 1878-1454; 1878-2434
• DOI: 10.1016/j.bbapap.2004.12.003

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, Ni 2+-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 2.3×10 4 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.