RESUMEN
The avidin-biotin technology has many applications, including molecular detection; immobilization; protein purification; construction of supramolecular assemblies and artificial metalloenzymes. Here we present the recombinant expression of novel biotin-binding proteins from bacteria and the purification and characterization of a secreted burkavidin from the human pathogen Burkholderia pseudomallei. Expression of the native burkavidin in Escherichia coli led to periplasmic secretion and formation of a biotin-binding, thermostable, tetrameric protein containing an intra-monomeric disulphide bond. Burkavidin showed one main species as measured by isoelectric focusing, with lower isoelectric point (pI) than streptavidin. To exemplify the potential use of burkavidin in biotechnology, an artificial metalloenzyme was generated using this novel protein-scaffold and shown to exhibit enantioselectivity in a rhodium-catalysed hydrogenation reaction.
Asunto(s)
Proteínas Bacterianas/metabolismo , Biotina/metabolismo , Proteínas Portadoras/metabolismo , Hidrogenasas/metabolismo , Proteínas Recombinantes/metabolismo , Estreptavidina/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Burkholderia pseudomallei/química , Proteínas Portadoras/química , Proteínas Portadoras/genética , Catálisis , Clonación Molecular , Estabilidad de Enzimas , Escherichia coli , Humanos , Hidrogenasas/química , Hidrogenasas/genética , Focalización Isoeléctrica , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Rodio/metabolismo , EstereoisomerismoRESUMEN
The mode of action of precious metal anticancer metallodrugs is generally believed to involve DNA as a target. However, the poor specificity of such drugs often requires high doses and leads to undesirable side-effects. With the aim of improving the specificity of a ruthenium piano-stool complex towards DNA, we employed a presenter protein strategy based on the biotin-avidin technology. Guided by the X-ray structure of the assembly of streptavidin and a biotinylated piano-stool, we explored the formation of metallodrug-mediated ternary complexes with the presenter protein and DNA. The assemblies bound more strongly to telomere G-quadruplexes than to double-stranded DNA; chemo-genetic modifications (varying the complex or mutating the protein) modulated binding to these targets. We suggest that rational targeting of small molecules by presenter proteins could be exploited to bind metallodrugs to preferred macromolecular targets.
Asunto(s)
ADN/química , Compuestos Organometálicos/química , Proteínas/química , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , ADN/efectos de los fármacos , ADN/metabolismo , Diseño de Fármacos , G-Cuádruplex/efectos de los fármacos , Estructura Molecular , Compuestos Organometálicos/síntesis química , Unión Proteica , Proteínas/metabolismo , Rutenio/química , Estreptavidina/químicaAsunto(s)
Alquenos/química , Tetróxido de Osmio/química , Estreptavidina/química , Sustitución de Aminoácidos , Sitios de Unión , Biocatálisis , Cristalografía por Rayos X , Dioxigenasas/genética , Dioxigenasas/metabolismo , Hidroxilación , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Estructura Terciaria de Proteína , EstereoisomerismoAsunto(s)
Evolución Molecular Dirigida , Hidrogenasas/química , Hidrogenasas/metabolismo , Catálisis , Cristalografía por Rayos X , Hidrogenasas/genética , Hidrogenasas/aislamiento & purificación , Modelos Moleculares , Estructura Molecular , Mutación/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Estereoisomerismo , Especificidad por SustratoRESUMEN
Artificial metalloenzymes result from the introduction of a catalytically competent non-native metal cofactor within a protein environment. In the present contribution, we summarize the recent achievements in the design and the optimization of such protein-based hybrid catalysts, with an emphasis on enantioselective transformations. The second part outlines the milestones required to achieve en masse production, screening and directed evolution of artificial metalloenzymes. In the spirit of Darwinian evolution, this will allow the full potential of such protein-based hybrid catalysts to be fully unraveled, thus complementing both homogeneous and enzymatic catalysis.