Sortase Conjugation

The produced scFv_VCAMV5-LPETG-His₈ was conjugated to G-BCN using the enzymatic sortase conjugation. Sortase enzyme was produced in-house as described previously.34 10 μM scFv_VCAMV5-LPETG-His₈, 30 μM sortase enzyme, and 300 μM G-BCN were reacted together in 50 mM Tris, 150 mM NaCl, 0.5 mM CaCl₂ (pH 8) at 37°C for 4 hr with shaking. Excess sortase and unreacted scFv was then removed by passing through a QIAGEN Ni-NTA Superflow column (1 mL). Excess G-BCN was subsequently removed by dialysis against PBS in a 10-kDa MWCO membrane.