The expression, purification and initial characterization of VCN as an active disintegrin.

<p><b>Panel A –</b> The production of Trx-VCN was assessed in different <i>E. coli</i> strains that were transformed, grown, induced, and processed under identical conditions. The same amount (5 µl) of cell lysates from each induced strain was loaded on a precast gel and Coomassie stained. Unlike the BL21 (DE3) strain, the lysates from both AD494 (DE3) and Origami B (DE3) strains generate a unique and consistent Trx-VCN band (shown by the arrow). By employing a modified media recipe, the Origami B (DE3) Trx-VCN transformants achieve higher cell densities at the end of the induction time, generating up to 200 mg of soluble VCN per L of bacterial culture after purification. <b>Panel B –</b> Coomassie stained gel showing the migration of Trx-VCN before and after TEV proteolysis (lanes 2 and 3, respectively) versus C18 reverse phase-HPLC purified VCN (lane 4). <b>Panel C </b><b>–</b> VCN and native CN exhibit an almost identical dose-dependent inhibitory effect against ADP-induced platelet aggregation when incubated with human platelet-rich plasma (with a calculated IC₅₀ of ∼60 nM). In contrast, the rCN construct, which is also expressed as a soluble polypeptide in Origami B (DE3), shows no inhibitory activity. <b>Panel D </b><b>–</b> The agonistic activity of VCN (FAK activation) was assessed in serum-starved non-migratory MDA-MB-435 cells kept in suspension and exposed to increasing concentrations of disintegrins for 30 min. Similar to dimeric CN, VCN is also shown to engage integrins agonistically (outside-in signaling).