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Objective·To investigate the evolution of sarcoma antigen 1 (SAGE1), a member of the cancer-testis antigen (CTA) family, in three representative primate species--Macaca mulatta (Rhesus), Callithrix jacchus (Marmoset), and Microcebus murinus (Mouse Lemur), as well as the conservation of its interaction with INTS3, a subunit of the integrator complex. Methods·The interaction between INTS3 and SAGE1 in these primates and the interaction between INTS3 and human SAGE1 mutants were first validated in HEK293T cells by using co-immunoprecipitation (Co-IP). Truncated proteins were then tagged with His-SUMO and GST, respectively, and expressed in Escherichia coli, followed by a series of purification steps to obtain the target proteins. Surface plasmon resonance (SPR) was used to measure the binding affinity of the target proteins, and size exclusion chromatography with multi-angle light scattering (SEC-MALS) was used to determine the stoichiometry of the complex. Additionally, molecular docking was employed to predict the binding model of the truncated SAGE1 and INTS3. Mutations were performed on human SAGE1 key interface residues to analyze their binding to INTS3 by Co-IP. Results·The interaction between the full-length human-derived INTS3 and the full-length SAGE1 from the three primate species was confirmed by Co-IP. Truncated proteins were purified through multiple steps of tandom chromatography. The dissociation constants of the three pairs of truncated INTS3-SAGE1 were measured via SPR, and the SEC-MALS results demonstrated that the binding ratio of INTS3-SAGE1 was 2∶1. Furthermore, molecular docking predicted a structural model of the truncated INTS3-SAGE1 complex and the binding interface was extensively constituted with hydrophobic contacts assisted by some hydrophilic interactions. The interaction between the mutant SAGE1 and INTS3 fulllength protein was significantly weakened. Conclusion·There is a conserved interaction between INTS3 and SAGE1 across Rhesus, Marmoset, and Mouse lemur. [ABSTRACT FROM AUTHOR]