Purification of the Eukaryotic Chaperonin CCT from Native Tissues

The eukaryotic CCT (chaperonin containing TCP1; also known as TRiC) is an essential molecular chaperone involved in the folding of nearly 10 % of newly synthesized proteins, including key cytoskeletal components such as actin and tubulin. It belongs to the Hsp60 family (60 kDa Heat Shock Proteins) or chaperonins, and it is composed of two stacked rings formed by eight different subunits. The CCT structural complexity makes its heterologous expression and purification highly challenging. To circumvent these difficulties, native purification from tissue remains a reliable approach. This protocol details the purification of bovine CCT from bull testis, a tissue naturally enriched in the complex due to its role in spermatogenesis. Originally developed by Frydman et al. in 1992 and extensively optimized by Prof. José María Valpuesta's group at CNB-CSIC over the past 25 years, the protocol has enabled key advances, including the crystallization and structural determination of bovine CCT. It continues to serve as a foundational tool for biochemical and structural studies of this essential chaperonin.