OVERPRODUCTION, PURIFICATION AND CHARACTERIZATION OF GROES AND GROEL FROM THERMOPHILIC BACILLUS-STEAROTHERMOPHILUS

To facilitate purification of the two chaperonins GroES and GroEL enco ded by the thermophilic Bacillus stearothermophilus, an Escherichia co li strain was constructed in which the groESL operon was replaced by t hat of B. stearothermophilus. This strain is perfectly viable, demonst rating that the B. stearothermophilus operon is functionally interchan geable with that of E. coli. To increase the amount of GroES, the groE S gene was fused to an IPTG-inducible promoter. Both proteins GroES an d GroEL were purified from E. coli using the standard protocol with so me modifications. This method should be applicable in all cases where a foreign groE operon can substitute that of E. coli. A preliminary ch aracterization of GroEL revealed that it has the same secondary struct ural elements as the E. coli homologue, but its thermodynamic stabilit y is significantly increased.