Thermus thermophilus encodes an archaeal-like fructose-1,6-bisphosphatase: Purification of native and recombinant protein for structural studies

  • Soulimane T
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Abstract

Fructose-1,6-bisphosphatase is one of the key enzymes of the gluconeogenic pathway. It catalyses the hydrolysis of fructose-1,6-bisphosphate to fructose-6-phosphate and inorganic phosphate. Fructose-1,6-bisphosphatase from the extreme thermophilic bacterium Thermus thermophilus has been purified by crystallisation approach. The final well-shaped crystals have been obtained using vapour diffusion sitting-drops in the presence of PEG 400 as the precipitating agent. The initially obtained native twinned crystals diffracted up to 1.2 resolution. Untwinned crystals used for data collection, however, were grown in the presence of thiomersal. They diffract to 1.8 resolution and belong to the space groups I422 with cell dimensions (i) a = b = 108.8 , c = 336.3 showing two molecules in the asymmetric unit, and (ii) a = b = 113.7 , c = 151.0 with one molecule in the asymmetric unit. The crystal structure has been solved by single anomalous dispersion using a 1.9 resolution. For further biochemical and biophysical investigations recombinant fructose-1,6-bisphosphatase has been produced in Escherichia coli. Both native (dissolved crystals) and recombinant material have been characterised by SDS-PAGE, N-terminal sequencing and MALDI-MS.© 2010 Elsevier Inc. All rights reserved.

Author-supplied keywords

  • Crystallogenesis
  • Fructose-1,6-bisphosphatase
  • Gluconeogenic pathway
  • Protein production
  • Thermus thermophilus

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Authors

  • T. Soulimane

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