Expression and characterization of a cytosolic glucose 6 phosphate dehydrogenase isoform from barley (Hordeum vulgare) roots.
Protein Expr Purif
; 112: 8-14, 2015 Aug.
Article
en En
| MEDLINE
| ID: mdl-25888782
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.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Hordeum
/
Clonación Molecular
/
Raíces de Plantas
/
Glucosafosfato Deshidrogenasa
/
NADP
Idioma:
En
Revista:
Protein Expr Purif
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2015
Tipo del documento:
Article
Pais de publicación:
Estados Unidos