Characterization of recombinant human protein C inhibitor expressed in Escherichia coli

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

• Publisher: Elsevier Science
• Year: 2005
• Tongue: English
• Weight: 203 KB
• Volume: 1748
• Category: Article
• ISSN: 1570-9639
• DOI: 10.1016/j.bbapap.2004.12.003

✦ Synopsis

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 2.3Â10 4 M À1 min À1 , respectively, and the inhibition rates were accelerated 25-, 71-, and 265-fold in the presence of 10 Ag/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.