Improving solubility of NR2B amino-terminal domain of N-methyl-d-aspartate receptor expressed in Escherichia coli.
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 (1mg/ml) exists as monodispersed species at 25 degrees 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 (64nM) and presence (116nM) 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.
Ng
Fui-Mee
FM
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.
Soh
Wanqin
W
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.
Geballe
Matthew T
MT
Department of Chemistry, Emory University, Atlanta, GA, USA.
Low
Chian-Ming
CM
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; Neurobiology Programme, Office of Life Sciences, National University of Singapore, Singapore. Electronic address: phclowcm@nus.edu.sg.
D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D058619:Maltose-Binding Proteins; D010880:Piperidines / Q000494:pharmacology; D011485:Protein Binding; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000737:chemistry* / Q000502:physiology; D012542:Scattering, Radiation; D012995:Solubility; D013696:Temperature
D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; C121001:NR2B NMDA receptor; D010880:Piperidines; D016194:Receptors, N-Methyl-D-Aspartate; D005982:Glutathione Transferase; C010739:ifenprodil