Recombinant expression of biologically active murine soluble EPCR.
Endothelial cell protein C receptor (EPCR) downregulates the coagulation system and prevents thrombosis by binding to protein C/activated protein C (APC) and factor VII/activated factor VII (VIIa). Recombinant APC and factor VIIa have been shown to be useful in a variety of clinical conditions. Murine models could prove extremely helpful in order to study in vivo actions of these drugs. It is therefore crucial to demonstrate the interaction between these and murine EPCR. We expressed the extracellular region of the murine EPCR in a yeast expression system and obtained a colony of Pichia pastoris that produce high amounts of recombinant extracellular murine EPCR, which we purified by liquid chromatography to homogeneity. The analysis of the interaction of EPCR with APC and factor VIIa was carried out using surface plasmon resonance technology. Murine EPCR binds to APC and factor VIIa with similar affinity than human EPCR. As for human EPCR, the binding is Ca2+ dependent while Mg2+ ions optimize it. In conclusion, we succeeded in establishing a system to produce enough recombinant soluble murine EPCR to perform biochemical studies. Murine EPCR binds to human APC and factor VIIa, which opens up new possibilities for characterizing the in vivo effect of APC and factor VII by using murine models.
López-Sagaseta
Jacinto
J
Division of Cardiovascular Sciences, Laboratory of Thrombosis and Haemostasis, Centre for Applied Medical Research, University of Navarra, Avenida Pio XII 55, 31008 Pamplona, Spain.
Montes
Ramón
R
Hermida
José
J
D000818:Animals; D001665:Binding Sites; D001779:Blood Coagulation Factors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002853:Chromatography, Liquid; D015942:Factor VIIa / Q000378:metabolism; D006801:Humans; D051379:Mice; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011486:Protein C / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D001779:Blood Coagulation Factors; D011486:Protein C; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C091963:activated protein C receptor; D015942:Factor VIIa