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Cloning, Expression, Purification and Characterization of His-Tagged Human Glucose-6-Phosphate Dehydrogenase: A Simplified Method for Protein Yield

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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.

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Abbreviations

G6PD:

Glucose-6-phosphate dehydrogenase

IMAC:

Immobilized metal affinity chromatography

LB:

Luria-Bertani

TB:

Terrific Broth

TEVP:

Tobacco etch virus protease

IPTG:

Isopropyl-1-thio-β-d-galactopyranoside

G6P:

Glucose-6-phosphate

CD:

Circular dichroism

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Acknowledgments

SGM is supported by Consejo Nacional de Ciencia y Tecnología Grant 154570. JOH is supported by Consejo Nacional de Ciencia y Tecnología Grant 106126. HRV is supported by Consejo Nacional de Ciencia y Tecnología Grant 104841. The technical assistance of Carmen Ortiz is greatly appreciated.

Conflict of interest

The authors declare no conflict of interest.

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Authors and Affiliations

  1. 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

    Saúl Gómez-Manzo, Jessica Terrón-Hernández, Ignacio de la Mora-de la Mora, Itzhel García-Torres, Gabriel López-Velázquez, Horacio Reyes-Vivas & Jesús Oria-Hernández

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  1. Saúl Gómez-Manzo
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  2. Jessica Terrón-Hernández
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  3. Ignacio de la Mora-de la Mora
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  4. Itzhel García-Torres
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  5. Gabriel López-Velázquez
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  6. Horacio Reyes-Vivas
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  7. Jesús Oria-Hernández
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Correspondence to Jesús Oria-Hernández.

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Gómez-Manzo, S., Terrón-Hernández, J., de la Mora-de la Mora, I. et al. Cloning, Expression, Purification and Characterization of His-Tagged Human Glucose-6-Phosphate Dehydrogenase: A Simplified Method for Protein Yield. Protein J 32, 585–592 (2013). https://doi.org/10.1007/s10930-013-9518-x

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