title: Cloning, expression, purification and characterization of his-tagged human glucose-6-phosphate dehydrogenase: a simplified method for protein yield.
abstract: Glucose-6-phosphate dehydrogenase (G6PD) catalyzes the first step of the pentose phosphate pathway. In erythrocytes, the functionality of the pathway is crucial to protect these cells against oxidative damage. G6PD deficiency is the most frequent enzymopathy in humans with a global prevalence of 4.9 %. The clinical picture is characterized by chronic or acute hemolysis in response to oxidative stress, which is related to the low cellular activity of G6PD in red blood cells. The disease is heterogeneous at genetic level with around 160 mutations described, mostly point mutations causing single amino acid substitutions. The biochemical studies aimed to describe the detrimental effects of mutations on the functional and structural properties of human G6PD are indispensable to understand the molecular physiopathology of this disease. Therefore, reliable systems for efficient expression and purification of the protein are highly desirable. In this work, human G6PD was heterologously expressed in Escherichia coli and purified by immobilized metal affinity chromatography in a single chromatographic step. The structural and functional characterization indicates that His-tagged G6PD resembles previous preparations of recombinant G6PD. In contrast with previous protein yield systems, our method is based on commonly available resources and fully accessible laboratory equipment; therefore, it can be readily implemented.
authors.affiliation: Laboratorio de Bioquímica Genética, Instituto Nacional de Pediatría, Secretaria de Salud, Insurgentes Sur 3700-C, Col. Insurgentes Cuicuilco, Delegación Coyoacán, 04530, Mexico, D.F., Mexico.
authors.forename: Saúl
authors.initials: S
authors.lastname: Gómez-Manzo
authors.forename: Jessica
authors.initials: J
authors.lastname: Terrón-Hernández
authors.forename: Ignacio
authors.initials: I
authors.lastname: de la Mora-de la Mora
authors.forename: Itzhel
authors.initials: I
authors.lastname: García-Torres
authors.forename: Gabriel
authors.initials: G
authors.lastname: López-Velázquez
authors.forename: Horacio
authors.initials: H
authors.lastname: Reyes-Vivas
authors.forename: Jesús
authors.initials: J
authors.lastname: Oria-Hernández
mesh_terms: D002846:Chromatography, Affinity; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D006639:Histidine; D006801:Humans; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D006639:Histidine; D005954:Glucosephosphate Dehydrogenase