Journal
PROTEIN EXPRESSION AND PURIFICATION
Volume 112, Issue -, Pages 8-14Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2015.03.016
Keywords
G6PDH; Barley; Pentose phosphate pathway; Anion exchange chromatography; pET3d; NADPH; Competitive inhibition
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Funding
- Project FORGIARE (FORmazione GIovAni alla RicErca) by Compagnia di San Paolo [10/FORG/ST/2012/5]
- Legge Regionale della Campania [5/2002]
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In plant cells, glucose 6 phosphate dehydrogenase (G6PDH-EC 1.1.1.49) regulates the oxidative pentose phosphate pathway (OPPP), a metabolic route involved in the production of NADPH for various biosynthetic processes and stress response. In this study, we report the overexpression of a cytosolic G6PDH isoform from barley (Hordeum vulgare) roots in bacteria, and the biochemical characterization of the purified recombinant enzyme (HvCy-G6PDH). A full-length cDNA coding for a cytosolic isoform of G6PDH was isolated, and the sequence was cloned into pET3d vector; the protein was overexpressed in Escherichia coli BL21 (DE3) and purified by anion exchange and affinity chromatography. The kinetic properties were calculated: the recombinant HvCy-G6PDH showed KMs and KINADPH comparable to those observed for the enzyme purified from barley roots; moreover, the analysis of NADPH inhibition suggested a competitive mechanism. Therefore, this enzyme could be utilised for the structural and regulatory characterization of this isoform in higher plants. (C) 2015 Elsevier Inc. All rights reserved.
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