Heterologous high-level E. coli expression, purification and biophysical characterization of the spine-associated RapGAP (SPAR) PDZ domain.
Spine-associated RapGAP (SPAR) is a 1783 residue, multidomain scaffolding protein which is a component of the NMDA receptor/PSD-95 complex in the post-synaptic density (PSD) of dendritic spines. Using a parallel expression screening approach, we identified a strategy to solubly express the SPAR PDZ domain in Escherichia coli. We show that maltose binding protein is required for the production of solubly expressed protein. We also show that small changes in construct length (2-5 residues) result in differential susceptibilities of the expressed proteins to proteolytic digestion, required for the expression tag removal. This has allowed us to identify a large-scale E. coli expression and purification protocol that results in the production of mg quantities of the SPAR PDZ domain. This is the first time that any of the multiple SPAR functional domains have been expressed in E. coli in quantities suitable for biophysical and biochemical studies, allowing us to investigate the role of the PDZ domain in SPAR function within the PSD.
Brown
Breann L
BL
Molecular Pharmacology and Physiology, Brown University, Providence, RI 02912, USA.
Hadley
Michael
M
Page
Rebecca
R
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D058619:Maltose-Binding Proteins; D019906:Nuclear Magnetic Resonance, Biomolecular; D054731:PDZ Domains; D017510:Protein Folding; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D002352:Carrier Proteins; D020690:GTPase-Activating Proteins; D058619:Maltose-Binding Proteins; D016194:Receptors, N-Methyl-D-Aspartate; C479629:Sipa1l1 protein, rat