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|Title:||Improving solubility of NR2B amino-terminal domain of N-methyl-d-aspartate receptor expressed in Escherichia coli|
Maltose binding protein
|Citation:||Ng, F.-M., Soh, W., Low, C.-M., Geballe, M.T. (2007). Improving solubility of NR2B amino-terminal domain of N-methyl-d-aspartate receptor expressed in Escherichia coli. Biochemical and Biophysical Research Communications 362 (1) : 69-74. ScholarBank@NUS Repository. https://doi.org/10.1016/j.bbrc.2007.07.164|
|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 (6×His-, 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 °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 6×His-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. © 2007 Elsevier Inc. All rights reserved.|
|Source Title:||Biochemical and Biophysical Research Communications|
|Appears in Collections:||Staff Publications|
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