Expression and Purification of Mammalian NMDA Receptor Protein for Functional Characterization

Methods Mol Biol. 2024:2799:13-27. doi: 10.1007/978-1-0716-3830-9_2.

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.

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

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Baculoviridae* / genetics
  • Chromatography, Affinity* / methods
  • Chromatography, Gel*
  • Gene Expression
  • Humans
  • Receptors, N-Methyl-D-Aspartate* / chemistry
  • Receptors, N-Methyl-D-Aspartate* / genetics
  • Receptors, N-Methyl-D-Aspartate* / isolation & purification
  • Receptors, N-Methyl-D-Aspartate* / metabolism
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Sf9 Cells

Substances

  • Receptors, N-Methyl-D-Aspartate
  • Recombinant Proteins