Dispersin-Mediated Fibrinogen and Vitronectin Degradation

Recombinant dispersin (1 μg in 100 μL of PBS – 10 μg/mL) or BSA were immobilized onto microplate wells. Purified MntC protein was used as positive control for fibrinogen cleavage (Salazar et al., 2014 (link)). Coated wells were blocked with 3% BSA diluted in PBS and plasminogen (10 μg/mL) was added. Incubation proceeded for 1 h at 37°C. After washing three times with PBS-T, human fibrinogen (10 μg) or human native vitronectin (1 μg) (Sigma-Aldrich) and plasminogen activator uPA (3U) were added. Reaction mixtures were incubated for 0, 1, or 4 h at 37°C, proteins were separated by 12% SDS-PAGE, and transferred to a nitrocellulose blotting membrane. Membranes were blocked with 5% skimmed milk for 1 h at room temperature with shaking, and the immunodetection was performed as described above for immunoblotting for dispersin detection, using primary antibodies anti-fibrinogen (Cloud-Clone, Katy, TX, United States) and anti-vitronectin (Complement Technology, Tyler, TX, United States) diluted 1:1000 and 1:5000, respectively, in 2.5% skimmed milk in PBS. Fibrinogen and vitronectin degradation products were visualized by ECL Analysis System (GE Healthcare) in the UVITEC Cambridge Image System (Alliance vs. 6.7).