title: Cloning, Expression and Characterization of Recombinant, NADH Oxidase from Giardia lamblia.
abstract: The NADH oxidase family of enzymes catalyzes the oxidation of NADH by reducing molecular O2 to H2O2, H2O or both. In the protozoan parasite Giardia lamblia, the NADH oxidase enzyme (GlNOX) produces H2O as end product without production of H2O2. GlNOX has been implicated in the parasite metabolism, the intracellular redox regulation and the resistance to drugs currently used against giardiasis; therefore, it is an interesting protein from diverse perspectives. In this work, the GlNOX gene was amplified from genomic G. lamblia DNA and expressed in Escherichia coli as a His-Tagged protein; then, the enzyme was purified by immobilized metal affinity chromatography, characterized, and its properties compared with those of the endogenous enzyme previously isolated from trophozoites (Brown et al. in Eur J Biochem 241(1):155-161, 1996). In comparison with the trophozoite-extracted enzyme, which was scarce and unstable, the recombinant heterologous expression system and one-step purification method produce a stable protein preparation with high yield and purity. The recombinant enzyme mostly resembles the endogenous protein; where differences were found, these were attributable to methodological discrepancies or artifacts. This homogenous, pure and functional protein preparation can be used for detailed structural or functional studies of GlNOX, which will provide a deeper understanding of the biology and pathogeny of G. lamblia.
authors.affiliation: Laboratorio de Bioquímica-Genética, Instituto Nacional de Pediatría, Secretaría de Salud. Av. Insurgentes Sur 3700 C, Insurgentes Cuicuilco, Delegación Coyoacán, 04530, Mexico, DF, Mexico.
authors.forename: Adriana
authors.initials: A
authors.lastname: Castillo-Villanueva
authors.affiliation: Laboratorio de Bioquímica-Genética, Instituto Nacional de Pediatría, Secretaría de Salud. Av. Insurgentes Sur 3700 C, Insurgentes Cuicuilco, Delegación Coyoacán, 04530, Mexico, DF, Mexico.
authors.forename: Sara Teresa
authors.initials: ST
authors.lastname: Méndez
authors.affiliation: Laboratorio de Bioquímica-Genética, Instituto Nacional de Pediatría, Secretaría de Salud. Av. Insurgentes Sur 3700 C, Insurgentes Cuicuilco, Delegación Coyoacán, 04530, Mexico, DF, Mexico.
authors.forename: Angélica
authors.initials: A
authors.lastname: Torres-Arroyo
authors.affiliation: Laboratorio de Bioquímica-Genética, Instituto Nacional de Pediatría, Secretaría de Salud. Av. Insurgentes Sur 3700 C, Insurgentes Cuicuilco, Delegación Coyoacán, 04530, Mexico, DF, Mexico. hreyesvivas@yahoo.com.mx.
authors.forename: Horacio
authors.initials: H
authors.lastname: Reyes-Vivas
authors.affiliation: Laboratorio de Bioquímica-Genética, Instituto Nacional de Pediatría, Secretaría de Salud. Av. Insurgentes Sur 3700 C, Insurgentes Cuicuilco, Delegación Coyoacán, 04530, Mexico, DF, Mexico. jesus.oria.inp@gmail.com.
authors.forename: Jesús
authors.identifier: http://orcid.org/0000-0001-7147-1879
authors.initials: J
authors.lastname: Oria-Hernández
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D016829:Giardia lamblia / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D010084:Oxidation-Reduction; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D009097:Multienzyme Complexes; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases
keywords: Drug resistance; Enzyme kinetics; Enzyme structure; Oxygen metabolism; Protein function; Redox regulation