Immobilization of Thrombin on Agarose-Based Supports for Affinity Tag Removal

Thrombin, a specific serine protease, is essential in recombinant protein purification by removing affinity tags. However, its soluble form has drawbacks like instability, contamination, and limited reusability. This study explores the covalent immobilization of bovine thrombin to enhance its performance as a reusable biocatalyst. Using the CapiPy tool, surface residues suitable for immobilization on agarose supports were identified. Thrombin immobilized on glyoxyl-activated agarose showed optimal results, efficiently removing 6xHis-tags from recombinant proteins with activity comparable to the soluble enzyme. It also cleaved other peptide tags, underscoring its versatility. It retained full activity after 1.5 h at 50 °C, while the soluble form was almost inactivated. The immobilized enzyme maintained consistent performance over 10 batch cycles and achieved a space-time yield of 4.7 g·L^-1·h^-1. These findings highlight the potential of immobilized thrombin as a robust and cost-effective tool to improving recombinant protein purification workflows, with significant implications for both industrial and research applications.