Development of an in vitro TurboID labeling assay for the detection of protoRAG-interacting proteins in the amphioxus

• Developed an in vitro TurboID proximity labeling assay to identify protoRAG-interacting proteins in amphioxus. • The assay uses recombinant TurboID-tagged BbRAG1L/BbRAG2L proteins incubated with amphioxus tissue lysates. • Biotinylated proteins are enriched and analyzed by LC-MS/MS, enabling the identification of candidate cofactors. • This method bypasses the need for transgenic amphioxus, facilitating studies on RAG evolution and regulation.