PURIFICATION AND CHARACTERIZATION OF CHAPERONIN-60 AND CHAPERONIN-10 FROM THE ANAEROBIC THERMOPHILE THERMOANAEROBACTER-BROCKII

Chaperonin 60 and chaperonin 10 (GroEL and GroES homologues, respectiv ely) have been isolated from extracts of the anaerobic thermophile The rmoanaerobacter brockii. A simple and rapid purification for chaperoni n 60 made use of hydrophobic and anion-exchange chromatographies, and could be readily scaled up; approximately 2 mg pure chaperonin 60 was obtained/g cells. In contrast with all other prokaryotic chaperonin 60 proteins that have been studied, which are tetradecamers, including t hose from Thermus sp., the T. brockii protein is a heptamer, and as is olated was not in association with chaperonin 10. The preparation is r eadily crystallized using 2-propanol or poly(ethylene glycol) with MgC l2. The N-terminal amino acid sequence of this preparation is similar to other thermophilic chaperonin 60 proteins. Chaperonin 10 was purifi ed from the flow-through of the first hydrophobic column (which bound chaperonin 60) using a more hydrophobic adsorbent to remove contaminat ing proteins, followed by anion-exchange chromatography. Chaperonin 10 was obtained with a yield of approximately 10% that of chaperonin 60. The subunit molecular mass of chaperonin 10 determined by electrospra y mass spectrometry is 10254 +/- 0.4Da, which is very similar to the m olecular mass of Escherichia coli GroES. Similarly, the subunit size o f chaperonin 60 determined by mass spectrometry is very similar to tha t of GroEL, at 57949 +/- 10 Da. T. brockii chaperonin 60 has an ATPase activity that is suppressed by chaperonin 10, and the two proteins to gether are active in protein-folding assays. Mitochondrial malate dehy drogenase was successfully refolded at 37 degrees C after denaturation in guanidine hydrochloride, using T. brockii chaperonin 60 and chaper onin 10, or chaperonin 60 and E. coli GroES. The denatured enzyme was protected from aggregation by association with chaperonin 60. Guanidin e-hydrochloride-denatured preparations of isocitrate dehydrogenase and secondary alcohol dehydrogenase isolated from T. brockii were also re folded at 60-65 degrees C. In each case, refolding required chaperonin 60, chaperonin 10 and ATP, giving up to 80% regeneration of control a ctivity.