Figure 2.

One-step synthesis of C to N protein fusions using the sortase-mediated swapping approach. (A) Schematic representation of synthesizing a C-to-N protein fusion via the sortase-swapping approach. (B) A nanobody equipped with an LPETG motif at its C terminus was fused to the GFP equipped with an LPETGGGGG at its N terminus via the sortase-swapping approach. LC-MS analyses confirmed the formation of the product. (C) SDS-PAGE was used to further confirm the formation of products after 2 h of reaction at 4 °C using immobilized sortase beads. (D) Flow cytometry analysis confirmed the functionality of the anti-human class II MHC nanobody after the synthesis of the nanobody–GFP fusion. Jurkat cells are class II MHC-negative, and Nalm6 cells are class II MHC-positive.