Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
2020939 | Protein Expression and Purification | 2010 | 6 Pages |
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 1.2Â Ã
resolution. Untwinned crystals used for data collection, however, were grown in the presence of thiomersal. They diffract to 1.8 Ã
resolution and belong to the space groups I422 with cell dimensions (i) a = b = 108.8 Ã
, c = 336.3 Ã
showing two molecules in the asymmetric unit, and (ii) a = b = 113.7 Ã
, c = 151.0 Ã
with one molecule in the asymmetric unit. The crystal structure has been solved by single anomalous dispersion using a 1.9Â Ã
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.
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Authors
T. Soulimane,