The recombinant thermosome from the hyperthermophilic archaeon Methanopyros kandleri: In vitro analysis of its chaperone activity

The archaeon Methanopyrus kandleri is the most thermophilic methanogen pres ently known. It contains a chaperonin (thermosome) which represents a 951 k Da homo-hexadecameric protein complex with NH4+-dependent ATPase activity, Since its synthesis is not increased upon heat shock, we set out to test it s chaperone function. In order to obtain the chaperonin in amounts sufficient for functional inve stigations, the gene encoding the 60 kDa subunit was expressed in E. coli B L21(DE3) cells. Purification yielded soluble, high-molecular-mass double-ri ng complexes, indistinguishable from the natural thermosome. in order to st udy the functional properties of the recombinant protein complex, pig citra te synthase, yeast alcohol dehydrogenase, yeast alpha-glucosidase, bovine i nsulin, and Thermotoga phosphoglycerate kinase were used as model substrate s. The results demonstrate that the recombinant M. kandleri thermosome possess es a chaperone-like activity in vitro, inhibiting aggregation as the major off-pathway-reaction during thermal unfolding and refolding of proteins aft er chemical denaturation. However, the chaperonin only forms dead-end compl exes with its non-native substrates, no release is detectable at temperatur es between 25 and 60 degrees C.