RESUMEN
Amyloid precursor protein (APP) cleaving enzyme (BACE) is the enzyme responsible for beta-site cleavage of APP, leading to the formation of the amyloid-beta peptide that is thought to be pathogenic in Alzheimer's disease (AD). Hence, BACE is an attractive pharmacological target, and numerous research groups have begun searching for potent and selective inhibitors of this enzyme as a potential mechanism for therapeutic intervention in AD. The mature enzyme is composed of a globular catalytic domain that is N-linked glycosylated in mammalian cells, a single transmembrane helix that anchors the enzyme to an intracellular membrane, and a short C-terminal domain that extends outside the phospholipid bilayer of the membrane. Here we have compared the substrate and active site-directed inhibitor binding properties of several recombinant constructs of human BACE. The constructs studied here address the importance of catalytic domain glycosylation state, inclusion of domains other than the catalytic domain, and incorporation into a membrane bilayer on the interactions of the enzyme active site with peptidic ligands. We find no significant differences in ligand binding properties among these various constructs. These data demonstrate that the nonglycosylated, soluble catalytic domain of BACE faithfully reflects the ligand binding properties of the full-length mature enzyme in its natural membrane environment. Thus, the use of the nonglycosylated, soluble catalytic domain of BACE is appropriate for studies aimed at understanding the determinants of ligand recognition by the enzyme active site.
Asunto(s)
Ácido Aspártico Endopeptidasas/química , Proteínas Recombinantes/química , Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide , Animales , Ácido Aspártico Endopeptidasas/metabolismo , Sitios de Unión , Células CHO , Catálisis , Dominio Catalítico , Línea Celular , Membrana Celular/metabolismo , Cromatografía Líquida de Alta Presión , Cricetinae , Relación Dosis-Respuesta a Droga , Drosophila , Endopeptidasas , Escherichia coli/metabolismo , Glicosilación , Humanos , Concentración 50 Inhibidora , Cinética , Ligandos , Luz , Membrana Dobles de Lípidos/metabolismo , Péptidos/química , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/metabolismo , Dispersión de Radiación , Factores de TiempoRESUMEN
Undesired adsorption of host cell proteins poses a big challenge for immobilized metal-ion affinity chromatography (IMAC) purification. In this study, by using His6-tagged protein Fab OPG C11 from Escherichia coli fermentation as a model, we found that the presence of low concentrations of EDTA-Mg2+ in feed streams weakens the adsorption but makes it more specific towards polyhistidine tag. By combining EDTA-Mg2+ treatment and periplasmic extraction, we developed a one-step purification procedure for His6-tagged recombinant Fab OPG C11 using Ni-IDA (iminodiacetic acid) chromatography. This procedure eliminated the buffer exchange step after periplasmic extraction, which is usually required before IMAC in order to remove EDTA. In addition to savings on time and cost, this procedure eliminates undesired adsorption of most host cell proteins thus significantly improves the purity of polyhistidine-tagged recombinant proteins. The strategy of EDTA-Mg2+ treatment may have general application potentials.
