Expression and Purification of Mammalian NMDA Receptor Protein for Functional Characterization.

Huang, Xuejing; Chen, Wanjin; Zhu, Shujia

Huang, Xuejing; Chen, Wanjin; Zhu, Shujia.

Afiliación

Huang X; Department of Neurology, The First Affiliated Hospital, Institute of Neuroscience, Fujian Key Laboratory of Molecular Neurology, Fujian Medical University, Fuzhou, Fujian, China.
Chen W; Institute of Neuroscience, State Key Laboratory of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai, China.
Zhu S; Department of Neurology, The First Affiliated Hospital, Institute of Neuroscience, Fujian Key Laboratory of Molecular Neurology, Fujian Medical University, Fuzhou, Fujian, China.

Methods Mol Biol ; 2799: 13-27, 2024.

Article en En | MEDLINE | ID: mdl-38727900

ABSTRACT

ABSTRACT

N-methyl-D-aspartate (NMDA) receptors are critical for brain function and serve as drug targets for the treatment of neurological and psychiatric disorders. They typically form the tetrameric assembly of GluN1-GluN2 (2A to 2D) subtypes, with their diverse three-dimensional conformations linked with the physiologically relevant function in vivo. Purified proteins of tetrameric assembled NMDA receptors have broad applications in the structural elucidation, hybridoma technology for antibody production, and high-throughput drug screening. However, obtaining sufficient quantity and monodisperse NMDA receptor protein is still technically challenging. Here, we summarize a paradigm for the expression and purification of diverse NMDA receptor subtypes, with detailed descriptions on screening constructs by fluorescence size-exclusion chromatography (FSEC), generation of recombinant baculovirus, expression in the eukaryotic expression system, protein purification by affinity chromatography and size-exclusion chromatography (SEC), biochemical and functional validation assays.

Asunto(s)

Baculoviridae; Cromatografía de Afinidad; Cromatografía en Gel; Receptores de N-Metil-D-Aspartato; Receptores de N-Metil-D-Aspartato/metabolismo; Receptores de N-Metil-D-Aspartato/genética; Receptores de N-Metil-D-Aspartato/aislamiento & purificación; Receptores de N-Metil-D-Aspartato/química; Animales; Baculoviridae/genética; Cromatografía de Afinidad/métodos; Humanos; Proteínas Recombinantes/aislamiento & purificación; Proteínas Recombinantes/genética; Proteínas Recombinantes/metabolismo; Expresión Génica; Células Sf9

Palabras clave

Affinity chromatography; Eukaryotic expression system; Fluorescence size-exclusion chromatography; NMDA receptors

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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Baculoviridae / Cromatografía de Afinidad / Cromatografía en Gel / Receptores de N-Metil-D-Aspartato Límite: Animals / Humans Idioma: En Revista: Methods Mol Biol Asunto de la revista: BIOLOGIA MOLECULAR Año: 2024 Tipo del documento: Article País de afiliación: China Pais de publicación: Estados Unidos

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