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

Journal Article · Fri Oct 12 04:00:00 EDT 2007 · Biochemical and Biophysical Research Communications

DOI:https://doi.org/10.1016/j.bbrc.2007.07.164· OSTI ID:21032922

Ng, F -M; Wanqin, Soh ^[1]; Geballe, Matthew T ^[2]; Low, C -M ^[1]

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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)

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.

• OSTI ID: 21032922
• Journal Information: Biochemical and Biophysical Research Communications, Journal Name: Biochemical and Biophysical Research Communications Journal Issue: 1 Vol. 362; ISSN 0006-291X; ISSN BBRCA9
• Country of Publication: United States
• Language: English

Related Subjects

60 APPLIED LIFE SCIENCES
DICHROISM
ESCHERICHIA COLI
GLUTATHIONE
LIGHT SCATTERING
MALTOSE
RECEPTORS