An engineered chaperonin caging a guest protein: Structural insights and potential as a protein expression tool

Abstract

The structure of a chaperonin caging a substrate protein is not quite clear. We made engineered group II chaperonins fused with a guest protein and analyzed their structural and functional features. Thermococcus sp. KS‐1 chaperonin α‐subunit (TCP) which forms an eightfold symmetric double‐ring structure was used. Expression plasmids were constructed which carried two or four TCP genes ligated head to tail in phase and a target protein gene at the 3′ end of the linked TCP genes. Electron microscopy showed that the expressed gene products with the molecular sizes of ∼120 kDa (di‐TCP) and ∼230 kDa (tetra‐TCP) formed double‐ring complexes similar to those of wild‐type TCP. The tetra‐TCP retained ATPase activity and its thermostability was significantly higher than that of the wild type. A 260‐kDa fusion protein of tetra‐TCP and green fluorescent protein (GFP, 27 kDa) was able to form the double‐ring complexes with green fluorescence. Image analyses indicated that the GFP moiety of tetra‐TCP/GFP fusion protein was accommodated in the central cavity, and tetra‐TCP/GFP formed the closed‐form similar to that crystallographically resolved in group II chaperonins. Furthermore, it was suggested that caging GFP expanded the cavity around the bottom. Using this tetra‐TCP fusion strategy, two virus structural proteins (21–25 kDa) toxic to host cells or two antibody fragments (25–36 kDa) prone to aggregate were well expressed in the soluble fraction of Escherichia coli . These fusion products also assembled to double‐ring complexes, suggesting encapsulation of the guest proteins. The antibody fragments liberated by site‐specific protease digestion exhibited ligand‐binding activities.