Cloning and overexpression in Escherichia coli of the gene encoding citrate synthase from the hyperthermophilic Archaeon Sulfolobus solfataricus
- 20 May 1998
- journal article
- research article
- Published by Springer Nature in Extremophiles
- Vol. 2 (2) , 61-66
- https://doi.org/10.1007/s007920050043
Abstract
The citrate synthase (CS) gene from the hyperthermophilic Archaeon Sulfolobus solfataricus has been cloned and sequenced. The gene encodes a polypeptide of 378 amino acids with a calculated polypeptide molecular mass of 42 679. High-level expression was achieved in Escherichia coli and the recombinant citrate synthase was purified to homogeneity using a heat step and dye-ligand affinity chromatography. This procedure yielded approximately 26 mg of pure CS per liter of culture, with a specific activity of 41 U/mg. The enzyme exhibited a half-life of 8 min at 95°C. A homology-modelled structure of the S. solfataricus CS has been generated using the crystal structure of the enzyme from the thermoacidophilic Archaeon Thermoplasma acidophilum with which it displays 58% sequence identity. The modelled structure is discussed with respect to the thermostability properties of the enzyme.Keywords
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