Characterization of recombinant human protein C inhibitor expressed in Escherichia coli.
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 x 10(4) M(-1) min(-1), respectively, and the inhibition rates were accelerated 25-, 71-, and 265-fold in the presence of 10 mug/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.
Réhault
Sophie M
SM
Department of Pathology and Laboratory Medicine, Carolina Cardiovascular Biology Center, The University of North Carolina, Chapel Hill, NC 27599-7035, USA.
Zechmeister-Machhart
Margareta
M
Fortenberry
Yolanda M
YM
Malleier
Julia
J
Binz
Nikki M
NM
Cooper
Scott T
ST
Geiger
Margarethe
M
Church
Frank C
FC
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D011486:Protein C / Q000037:antagonists & inhibitors / Q000378:metabolism; D018046:Protein C Inhibitor / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D015842:Serine Proteinase Inhibitors / Q000235:genetics / Q000378:metabolism*; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors / Q000378:metabolism
D011486:Protein C; D018046:Protein C Inhibitor; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D006493:Heparin; D013917:Thrombin; D014568:Urokinase-Type Plasminogen Activator