Cloning and overexpression in Escherichia coli of the gene encoding citrate synthase from the hyperthermophilic Archaeon Sulfolobus solfataricus

H. Connaris, S.M. West, D.W. Hough, M.J. Danson

Research output: Contribution to journalArticlepeer-review

15 Citations (Scopus)

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 42679. 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.

Original languageEnglish
Pages (from-to)61-66
Number of pages6
JournalExtremophiles
Volume2
Issue number2
DOIs
Publication statusPublished - 1 May 1998

Keywords

  • Thermophile
  • Citrate synthase
  • Thermostability
  • Archaea
  • Gene sequence
  • Sulfolobus

Fingerprint

Dive into the research topics of 'Cloning and overexpression in Escherichia coli of the gene encoding citrate synthase from the hyperthermophilic Archaeon Sulfolobus solfataricus'. Together they form a unique fingerprint.

Cite this