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

Autor: Furutani M; Sekisui Chemical Co. Ltd., Hyakuyama 2-1, Shimamoto-cho, Mishima-gun, Osaka 618-8589, Japan. furutani002@sekisui.jp, Hata J, Shomura Y, Itami K, Yoshida T, Izumoto Y, Togi A, Ideno A, Yasunaga T, Miki K, Maruyama T
Jazyk: angličtina
Zdroj: Protein science : a publication of the Protein Society [Protein Sci] 2005 Feb; Vol. 14 (2), pp. 341-50.
DOI: 10.1110/ps.041043905
Abstrakt: 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 alpha-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.
Databáze: MEDLINE
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