Author:
Hamedani Nasim Shahidi,Happich Felix Lucian,Klein Eva-Maria,Rühl Heiko,Mayer Günter,Oldenburg Johannes,Müller Jens,Pötzsch Bernd
Abstract
AbstractActivated protein C (APC) is a serine protease with anticoagulant and cytoprotective activities which make it an attractive target for diagnostic and therapeutic applications. In this work, we present one-step activation of APC from a commercial source of protein C (PC, Ceprotin) followed by rapid and efficient purification using an APC-specific aptamer, HS02-52G, loaded on MyOne superparamagnetic beads. Due to the Ca2+-dependent binding of APC to HS02-52G, an efficient capturing of APC was applied in the presence of Ca2+ ions, while a gentle release of captured APC was achieved in the elution buffer containing low EDTA concentration (5 mM). The captured and eluted APC showed more than 95% purity according to SDS-PAGE gel analysis and an enzyme-linked fluorescent assay (VIDAS Protein C). The purification yield of 45% was calculated when 4.2 µg APC was used, however this yield reduced to 21% if the starting amount of APC increased to 28.5 µg. Altogether, this method is recommended for rapid and efficient PC activation and APC purification. The purified APC can be used directly for downstream processes where high concentration of pure and active APC is needed.
Funder
Universitätsklinikum Bonn
Publisher
Springer Science and Business Media LLC
Reference43 articles.
1. Griffin, J. H., Fernandez, J. A., Gale, A. J. & Mosnier, L. O. Activated protein C. J. Thromb. Haemost. 5(Suppl 1), 73–80. https://doi.org/10.1111/j.1538-7836.2007.02491.x (2007).
2. Gruber, A. & Griffin, J. H. Direct detection of activated protein C in blood from human subjects. Blood 79, 2340–2348 (1992).
3. Okajima, K. et al. Effect of protein C and activated protein C on coagulation and fibrinolysis in normal human subjects. Thromb. Haemost. 63, 48–53 (1990).
4. Griffin, J. H., Zlokovic, B. V. & Mosnier, L. O. Protein C anticoagulant and cytoprotective pathways. Int. J. Hematol. 95, 333–345. https://doi.org/10.1007/s12185-012-1059-0 (2012).
5. Berg, J. M., Tymoczko, J. L. & Stryer, L. The purification of proteins is an essential first step in understanding their function. Biochemistry 5, 20 (2002).
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