• Developed an in vitro TurboID proximity labeling assay to identify protoRAG-interacting proteins in amphioxus, overcoming the lack of endogenous expression and transgenic challenges.
• The assay uses purified MBP-TurboID-BbRAG1L/2L fusion proteins incubated with amphioxus tissue lysates, followed by LC-MS/MS analysis, enabling detection of interaction partners.
• This approach provides a powerful tool to study the regulatory network of protoRAG and its evolutionary transition from transposase to recombinase.
• The method can be adapted to other RAG-like proteins and facilitates understanding of host coevolution and adaptive immunity origins.