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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
Author(s) -
Wu Huiping,
Bruley Duane F.
Publication year - 1999
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp9901015
Subject(s) - chemistry , chromatography , protein s , protein purification , affinity chromatography , blood proteins , elution , protein c , biochemistry , enzyme
Protein C (PC) is a natural anticoagulant and antithrombotic present in human blood at a concentration of 4 μg/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.