Chelator, metal ion and buffer studies for protein C separation

Protein C (PC) is the pivotal anticoagulant and antithrombotic in the human coagulation cascade. PC deficiency can result in major medical problems such as deep vein thrombosis (DVT), leading to tissue oxygen deprivation. PC treatment has no bleeding or skin necrosis problems because it circulates in the blood as a zymogen and is only activated when and where it is needed. One source of PC is transgenic animal milk. The major components in the milk. such as alpha-casein, beta-casein, kappa-cascin, alpha-lactalbumin and beta-lactoglobulin, are proteins that must be separated from PC. Immobilized metal affinity chromatography (IMAC) is an inexpensive separation technology with relatively high specificity, and it has great potential for difficult protein separations. After systematic studies of different chelator, metal ion and buffers, the combination of iminodiacetic acid (IDA) and Fe was found to be effective to separate PC from major milk components. a-Lactalbumin and P-lactoglobulin fell through the column in the starting buffer. PC was eluted. a-Casein, P-casein, K-casein remained bound in the column after PC elution. This technology might be applied for PC separation from transgenic animal milk. It is very important for PC production in large quantities and at low cost to treat PC-deficient patients. (C) 2002 Elsevier Science Inc. All rights reserved.