Homologous human blood protein separation using immobilized metal affinity chromatography: protein C separation from prothrombin with application to the separation of factor IX and prothrombin.
Protein C (PC) is a natural anticoagulant and antithrombotic present in human blood at a concentration of 4 microg/mL. Its deficiency can result in excessive clotting and thrombosis. Protein C can be obtained from human blood plasma; however, there are other coagulant proteins in blood, including prothrombin (factor II), which is present in relatively large amounts and is one of the most active components. Protein C and prothrombin are homologous proteins with similar biochemical features; therefore, immunoaffinity chromatography is used for their separation. However, this technology is very expensive, protein C recovery and activity is low, and contamination problems with mouse antibody are likely. Immobilized metal affinity chromatography (IMAC) utilizes the protein metal-binding properties for protein separation. Protein C has twelve surface-accessible histidines, which are the major metal-binding groups for IMAC separation. After investigating metal ion-binding properties of protein C, we used an IDA-Cu column to separate protein C and prothrombin. Following protein adsorption to the column, prothrombin was washed out using a sodium phosphate buffer containing 2 mM imidazole and protein C was recovered with 15 mM imidazole in the buffer. The mild elution condition allows a high protein C activity and a high recovery. Also, this technology introduces no immunoglobulins, and it is relatively inexpensive. IMAC could replace the immunoaffinity technology for the large-scale separation of protein C from blood plasma Cohn Fraction IV-1. In addition, this work demonstrates a significant application of this technology for the separation of factor IX from prothrombin. Prothrombin has proven to be a harmful contaminant in factor IX cocktails that have been administered to humans in the treatment of hemophilia B.
Wu
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Department of Chemical and Biochemical Engineering, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland 21250, USA.
Bruley
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D001798:Blood Proteins / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods*; D003300:Copper; D005164:Factor IX / Q000302:isolation & purification*; D006801:Humans; D011486:Protein C / Q000302:isolation & purification*; D011516:Prothrombin / Q000302:isolation & purification*
D001798:Blood Proteins; D011486:Protein C; D003300:Copper; D011516:Prothrombin; D005164:Factor IX