title: Expression of a soluble glycine binding domain of the NMDA receptor in Escherichia coli.
abstract: Glycine is an essential co-agonist of the excitatory N-methyl-D-aspartate (NMDA) receptor. The glycine binding site of this subtype of ionotropic glutamate receptors is formed by the S1 and S2 regions of the NR1 subunit. Here, different S1S2 fusion proteins were expressed and purified from Escherichia coli cultures, and refolding protocols were established allowing the production of 30 mg of soluble S1S2 fusion protein from 1 liter bacterial culture. After affinity purification and renaturation, two of the fusion proteins (S1S2 and S1S2-V1) bound the competitive glycine site antagonist [3H]MDL105,519 with K(d) values of 9.35 and 3.9 nM, respectively. In contrast, with three other constructs (S1S2M, S1S2-V2, and -V3) saturable ligand binding could not be obtained. These results redefine the S1S2 domains required for high-affinity glycine binding. Furthermore, our high-affinity binding proteins may be used for the large-scale production of the glycine binding core region for future structural studies.
authors.affiliation: Department of Anatomy and Cellular Neurobiology, University of Ulm, Albert-Einstein-Allee 11, D-89081 Ulm, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Neugebauer
authors.forename: Heinrich
authors.initials: H
authors.lastname: Betz
authors.forename: Jochen
authors.initials: J
authors.lastname: Kuhse
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005998:Glycine / Q000378:metabolism*; D007211:Indoles / Q000378:metabolism; D008970:Molecular Weight; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D016194:Receptors, N-Methyl-D-Aspartate / Q000737:chemistry* / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D012995:Solubility
chemical_list: D007211:Indoles; C101788:MDL 105519; D016194:Receptors, N-Methyl-D-Aspartate; D011993:Recombinant Fusion Proteins; D005998:Glycine