skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Improving solubility of NR2B amino-terminal domain of N-methyl-D-aspartate receptor expressed in Escherichia coli

Abstract

The amino-terminal domains (ATDs) of N-methyl-D-aspartate (NMDA) receptors contain binding sites for modulators and may serve as potential drug targets in neurological diseases. Here, three fusion tags (6xHis-, GST-, and MBP-) were fused to the ATD of NMDA receptor NR2B subunit (ATD2B) and expressed in Escherichia coli. Each tag's ability to confer enhanced solubility to ATD2B was assessed. Soluble ATD2B was successfully obtained as a MBP fusion protein. Dynamic light scattering revealed the protein (1 mg/ml) exists as monodispersed species at 25 {sup o}C. Functional studies using circular dichroism showed that the soluble MBP-ATD2B bound ifenprodil in a dose-dependent manner. The dissociation constants obtained for ifenprodil were similar in the absence (64 nM) and presence (116 nM) of saturating concentration of maltose. Moreover, the yield of soluble MBP-ATD2B is 18 times higher than the refolded 6xHis-ATD2B. We have reported a systematic comparison of three different affinity tagging strategies and identified a rapid and efficient method to obtain large amount of ATD2B recombinant protein for biochemical and structural studies.

Authors:
;  [1];  [2];  [1]
  1. Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Centre for Life Sciences 04-06, 28 Medical Drive, Singapore S117456 (Singapore)
  2. Department of Chemistry, Emory University, Atlanta, GA (United States)
Publication Date:
OSTI Identifier:
21032922
Resource Type:
Journal Article
Journal Name:
Biochemical and Biophysical Research Communications
Additional Journal Information:
Journal Volume: 362; Journal Issue: 1; Other Information: DOI: 10.1016/j.bbrc.2007.07.164; PII: S0006-291X(07)01642-7; Copyright (c) 2007 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA); Journal ID: ISSN 0006-291X
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; DICHROISM; ESCHERICHIA COLI; GLUTATHIONE; LIGHT SCATTERING; MALTOSE; RECEPTORS

Citation Formats

Ng, F -M, Wanqin, Soh, Geballe, Matthew T, Low, C -M, and Neurobiology Programme, Office of Life Sciences, National University of Singapore. Improving solubility of NR2B amino-terminal domain of N-methyl-D-aspartate receptor expressed in Escherichia coli. United States: N. p., 2007. Web. doi:10.1016/j.bbrc.2007.07.164.
Ng, F -M, Wanqin, Soh, Geballe, Matthew T, Low, C -M, & Neurobiology Programme, Office of Life Sciences, National University of Singapore. Improving solubility of NR2B amino-terminal domain of N-methyl-D-aspartate receptor expressed in Escherichia coli. United States. https://doi.org/10.1016/j.bbrc.2007.07.164
Ng, F -M, Wanqin, Soh, Geballe, Matthew T, Low, C -M, and Neurobiology Programme, Office of Life Sciences, National University of Singapore. Fri . "Improving solubility of NR2B amino-terminal domain of N-methyl-D-aspartate receptor expressed in Escherichia coli". United States. https://doi.org/10.1016/j.bbrc.2007.07.164.
@article{osti_21032922,
title = {Improving solubility of NR2B amino-terminal domain of N-methyl-D-aspartate receptor expressed in Escherichia coli},
author = {Ng, F -M and Wanqin, Soh and Geballe, Matthew T and Low, C -M and Neurobiology Programme, Office of Life Sciences, National University of Singapore},
abstractNote = {The amino-terminal domains (ATDs) of N-methyl-D-aspartate (NMDA) receptors contain binding sites for modulators and may serve as potential drug targets in neurological diseases. Here, three fusion tags (6xHis-, GST-, and MBP-) were fused to the ATD of NMDA receptor NR2B subunit (ATD2B) and expressed in Escherichia coli. Each tag's ability to confer enhanced solubility to ATD2B was assessed. Soluble ATD2B was successfully obtained as a MBP fusion protein. Dynamic light scattering revealed the protein (1 mg/ml) exists as monodispersed species at 25 {sup o}C. Functional studies using circular dichroism showed that the soluble MBP-ATD2B bound ifenprodil in a dose-dependent manner. The dissociation constants obtained for ifenprodil were similar in the absence (64 nM) and presence (116 nM) of saturating concentration of maltose. Moreover, the yield of soluble MBP-ATD2B is 18 times higher than the refolded 6xHis-ATD2B. We have reported a systematic comparison of three different affinity tagging strategies and identified a rapid and efficient method to obtain large amount of ATD2B recombinant protein for biochemical and structural studies.},
doi = {10.1016/j.bbrc.2007.07.164},
url = {https://www.osti.gov/biblio/21032922}, journal = {Biochemical and Biophysical Research Communications},
issn = {0006-291X},
number = 1,
volume = 362,
place = {United States},
year = {2007},
month = {10}
}