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

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 thetetrameric 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 theeukaryotic expression system, protein purification by affinity chromatography and size-exclusion chromatography (SEC), biochemical and functional validation assays.