Vx3 Protein Expression and Purification

Vx3 protein was expressed and purified based on our previous work.19 (link),20 (link) In brief, pUbiG101-Vx3(A7)-eGFP expressing plasmid was introduced into E. coli DH5-α competent cells (Invitrogen) to induce the expression of His-Vx3-eGFP fusion protein. Next, cells were harvested and treated with lysozyme (Sigma). After sonication and centrifugation, supernatant containing Vx3 proteins was purified using Ni-NTA agarose beads at 4°C overnight. Beads were washed 3 times with native washing buffer and the purified Vx3 protein was eluted by native elution buffer (Figure S1). The protein concentration of Vx3 was quantified by Bradford Protein Assay Kit (Beyotime Biotechnology) according to the manufacturer’s protocol.