title: Application of Strep-Tactin XT for affinity purification of Twin-Strep-tagged CB2, a G protein-coupled cannabinoid receptor.
abstract: Human cannabinoid receptor CB2belongs to the class A of G protein-coupled receptor (GPCR). CB2is predominantly expressed in membranes of cells of immune origin and is implicated in regulation of metabolic pathways of inflammation, neurodegenerative disorders and pain sensing. High resolution structural studies of CB2require milligram quantities of purified, structurally intact protein. While we previously reported on the methodology for expression of the recombinant CB2and its stabilization in a functional state, here we describe an efficient protocol for purification of this protein using the Twin-Strep-tag/Strep-Tactin XT system. To improve the affinity of interaction of the recombinant CB2with the resin, the double repeat of the Strep-tag (a sequence of eight amino acids WSHPQFEK), named the Twin-Strep-tag was attached either to the N- or C-terminus of CB2via a short linker, and the recombinant protein was expressed in cytoplasmic membranes of E. coli as a fusion with the N-terminal maltose binding protein (MBP). The CB2was isolated at high purity from dilute solutions containing high concentrations of detergents, glycerol and salts, by capturing onto the Strep-Tactin XT resin, and was eluted from the resin under mild conditions upon addition of biotin. Surface plasmon resonance studies performed on the purified protein demonstrate the high affinity of interaction between the Twin-Strep-tag fused to the CB2and Strep-Tactin XT with an estimated Kd in the low nanomolar range. The affinity of binding did not vary significantly in response to the position of the tag at either N- or C-termini of the fusion. The binding capacity of the resin was several-fold higher for the tag located at the N-terminus of the protein as opposed to the C-terminus- or middle of the fusion. The variation in the length of the linker between the double repeats of the Strep-tag from 6 to 12 amino acid residues did not significantly affect the binding. The novel purification protocol reported here enables efficient isolation of a recombinant GPCR expressed at low titers in host cells. This procedure is suitable for preparation of milligram quantities of stable isotope-labelled receptor for high-resolution NMR studies.
authors.affiliation: National Institute on Alcoholism and Alcohol Abuse, National Institutes of Health, 5625 Fishers Lane, Room 3N17, Rockville, MD, 20892, USA. Electronic address: yeliseeva@mail.nih.gov.
authors.forename: Alexei
authors.initials: A
authors.lastname: Yeliseev
authors.affiliation: National Institute on Alcoholism and Alcohol Abuse, National Institutes of Health, 5625 Fishers Lane, Room 3N17, Rockville, MD, 20892, USA.
authors.forename: Lioudmila
authors.initials: L
authors.lastname: Zoubak
authors.affiliation: IBA GmbH, Rudolf-Wissell-Str., 28, D-37079, Goettingen, Germany.
authors.forename: Thomas G M
authors.initials: TGM
authors.lastname: Schmidt
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D004926:Escherichia coli; D015870:Gene Expression; D006801:Humans; D019906:Nuclear Magnetic Resonance, Biomolecular; D043885:Receptor, Cannabinoid, CB2 / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D020349:Surface Plasmon Resonance
chemical_list: C588309:CNR2 protein, human; D043885:Receptor, Cannabinoid, CB2; D011993:Recombinant Fusion Proteins