Single-Step Purification of Endogenous Human Chaperonin CCT

The eukaryotic chaperonin CCT (chaperonin-containing TCP-1) is essential for protein homeostasis in eukaryotic cells, and its dysfunction has been linked to an increasing number of pathologies, including cancer, neurodegeneration, and infectious diseases. Traditional CCT purification methods are complex, multi-step processes, while newer affinity chromatography approaches rely on tagged, non-native CCT. Here, we describe a single-step method to purify endogenous CCT from a custom Expi293F cell line generated using the Sleeping Beauty transposon system. This cell line inducibly expresses a phosducin-like protein (PhLP1) with a C-terminal Twin-Strep-tag, which binds CCT with high affinity. Using Strep-Tactin XT resin, we isolate CCT directly from cell lysates via the PhLP1-CCT interaction, achieving high-purity CCT with yields comparable to traditional methods. This rapid, specific approach simplifies CCT purification while maintaining scalability and efficiency.