Herein we report the synthesis and isolation of a shape-persistent Janus protein nanoparticle derived from the biomolecular machine chaperonin GroEL (^AGroEL^B) and its application to DNA-mediated ternary supramolecular copolymerization. To synthesize ^AGroEL^B with two different DNA strands A and B at its opposite apical domains, we utilized the unique biological property of GroEL, i.e., Mg²⁺/ATP-mediated ring exchange between ^AGroEL^A and ^BGroEL^B with their hollow cylindrical double-decker architectures. This exchange event was reported more than 24 years ago but has never been utilized for molecular engineering of GroEL. We leveraged DNA nanotechnology to purely isolate Janus ^AGroEL^B and succeeded in its precision ternary supramolecular copolymerization with two DNA comonomers, A** and B*, that are partially complementary to A and B in ^AGroEL^B, respectively, and programmed to self-dimerize on the other side. Transmission electron microscopy allowed us to confirm the formation of the expected dual-periodic copolymer sequence −(^(B*/B)GroEL^(A/A**/A**/A)GroEL^(B/B*))– in the form of a laterally connected lamellar assembly rather than a single-chain copolymer
