PB1109 - Development and evaluation of an aptamer-based affinity purification method for activated protein C (APC)

Background: Activated protein C (APC) as one of the main anticoagulant pathways is a serine protease with anticoagulant and cytoprotective activities. To study the role of APC in cellular and molecular mechanisms, a reliable and perpetual source of APC is required. DNA aptamers are attractive capturing ligands for affinity purification since they are able to discriminate between active and zymogenic state of a protein. Combining the advantages of using aptamers as capturing ligands with magnetic beads for one-step purification allows rapid processing required for separation of the active enzyme APC.

Aims: The aim of this study was to develop a generation and purification method to access a perpetual source of the purified APC for diagnostic and clinical applications.

Methods: In this work, one-step thrombin-mediated activation of APC from a commercial source of protein C (PC, Ceprotin®) was followed by rapid and efficient purification of generated APC using the APC-specific aptamer HS02-52G chemically immobilized on MyOne® superparamagnetic beads.

Results: Due to the Ca2+-dependent binding manner 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 achieved using elution buffer containing 5 mM EDTA. In addition, using the MyOne® beads having smaller diameter (1 µm) showed more efficient purification capacity in comparison to M-270® magnetic beads (2.8 µm). The purification yield of 72.6% was calculated when 5 µg PC was activated, however this yield reduced to 11.5% using 20 µg PC starting amount. The captured and eluted APC showed 95% purity with no detectable co-purified PC using enzyme-linked fluorescence assay (VIDAS® Protein C).

Conclusion(s): Altogether, this method is recommended for rapid and efficient APC generation and purification with high purity and reasonable yield. The purified APC can be used directly for downstream processes where high concentration of pure and active APC is needed.