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The Recombinant Thermosome from the Hyperthermophilic Archaeon Methanopyrus kandleri: In Vitro Analysis of Its Chaperone Activity
Citation Information: Biological Chemistry. Volume 380, Issue 1, Pages 55–62, ISSN (Print) 1431-6730, DOI: 10.1515/BC.1999.007, June 2005
- Published Online:
The archaeon Methanopyrus kandleri is the most thermophilic methanogen presently known. It contains a chaperonin (thermosome) which represents a 951 kDa homo-hexadecameric protein complex with NH4 +-dependent ATPase activity. Since its synthesis is not increased upon heat shock, we set out to test its chaperone function.
In order to obtain the chaperonin in amounts sufficient for functional investigations, the gene encoding the 60 kDa subunit was expressed in E. coli BL21(DE3) cells. Purification yielded soluble, high-molecularmass double-ring complexes, indistinguishable from the natural thermosome. In order to study the functional properties of the recombinant protein complex, pig citrate synthase, yeast alcohol dehydrogenase, yeast α-glucosidase, bovine insulin, and Thermotoga phosphoglycerate kinase were used as model substrates.
The results demonstrate that the recombinant M. kandleri thermosome possesses a chaperone-like activity in vitro, inhibiting aggregation as the major off-pathway-reaction during thermal unfolding and refolding of proteins after chemical denaturation. However, the chaperonin only forms dead-end complexes with its non-native substrates, no release is detectable at temperatures between 25 and 60 °C.
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