CLONING AND OVEREXPRESSION IN ESCHERICHIA-COLI OF THE GENE ENCODING CITRATE SYNTHASE FROM THE HYPERTHERMOPHILIC ARCHAEON SULFOLOBUS-SOLFATARICUS

The citrate synthase (CS) gene from the hyperthermophilic Archaeon Sul folobus solfataricus has been cloned and sequenced. The gene encodes a polypeptide of 375 amino acids with a calculated polypeptide molecula r mass of 42679. High-level expression was achieved in Escherichia col i and the recombinant citrate synthase was purified to homogeneity usi ng a heat step and dye-ligand affinity chromatography. This procedure yielded approximately 26 mg of pure CS per liter of culture, with a sp ecific activity of 41 U/mg. The enzyme exhibited a half-life of 8 min at 95 degrees C. A homology-modelled structure of the S. solfataricus CS has been generated using the crystal structure of the enzyme from t he thermoacidophilic Archaeon Thermoplasma acidophilum with which it d isplays 58% sequence identity. The modelled structure is discussed wit h respect to the thermostability properties of the enzyme.