Open AccessRecombinant homo‐ and hetero‐oligomers of an ultrastable chaperonin from the archaeon Pyrodictium occultum show chaperone activity in vitro
Author(s) -
Minuth T.,
Frey G.,
Lindner P.,
Rachel R.,
Stetter K. O.,
Jaenicke R.
Publication year - 1998
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.1998.2580837.x
Subject(s) - recombinant dna , circular dichroism , protein quaternary structure , escherichia coli , molecular mass , biochemistry , chaperone (clinical) , ultracentrifuge , size exclusion chromatography , thermophile , chemistry , chaperonin , biophysics , biology , protein folding , protein subunit , enzyme , medicine , pathology , gene
The archaeon Pyrodictium occultum is one of the most thermophilic organisms presently known. Previous experiments provided support for the significant contribution of a high‐molecular‐mass protein complex to the extreme thermotolerance of P. occultum . This protein complex, the ’thermosome', is composed of two subunits, α and β, which form a hexadecameric double ring complex. In order to obtain the thermosome in amounts sufficient for structural and functional investigations, we produced the two subunits jointly and separately in Escherichia coli BL21(DE3). In all three cases, we isolated soluble, high‐molecular‐mass double‐ring complexes from E. coli BL21(DE3). On electron micrographs, the recombinant complexes were indistinguishable from each other and from the natural thermosome. To characterize the quaternary structure of the recombinant particles, we used native gel electrophoresis, analytical gel filtration, and analytical ultracentrifugation. Spectral analysis, using absorption, fluorescence emission and far‐UV circular dichroism spectroscopy were applied to compare the three recombinant protein complexes with the natural thermosome from P. occultum . All three recombinant complex species exhibit ATPase activity. Furthermore, we could demonstrate that the recombinant complexes slow down the aggregation of citrate synthase, alcohol dehydrogenase, and insulin. Thus, we conclude that the recombinant protein complexes exhibit a chaperone‐like activity, interacting with non‐native proteins; they do so at temperatures far below the lower physiological limit of growth.