H. Connaris et al., CLONING AND OVEREXPRESSION IN ESCHERICHIA-COLI OF THE GENE ENCODING CITRATE SYNTHASE FROM THE HYPERTHERMOPHILIC ARCHAEON SULFOLOBUS-SOLFATARICUS, Extremophiles, 2(2), 1998, pp. 61-66
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.