The human peripheral cannabinoid receptor (CB2) was expressed as a fusion with the maltosebinding protein (at the N-terminus), thioredoxin A (at the C-terminus) and two small affinity tags (a Strep-tag and a polyhistidine tag). Expression levels of the recombinant receptor in Escherichia coli BL21(DE3) cells were dependent on location and type of tags in the expression construct, and were as high as 1-2 mg per liter of bacterial culture. The recombinant receptor was ligand bindingcompetent, and activated cognate G-proteins in an in vitro coupled assay. The fusion CB2-125 protein was purified by immobilized metal affinity chromatography on a Ni-NTA resin. Maltose-binding protein, thioredoxin and a decahistidine tag were removed from the fusion by treatment with Tobacco etch virus (Tev) protease. Purification to over 90% homogeneity of the resulting CB2, containing an N-terminal Strep-tag was achieved by affinity chromatography on a StrepTactin resin. Circular dichroism spectroscopy indicated an α-helical content of the purified recombinant protein of ∼54%. The expression and purification protocol allows for production of large (milligram) quantities of functional peripheral cannabinoid receptor, suitable for subsequent structural characterization. Preliminary results of reconstitution experiments indicate that the CB2 has retained its ligand-binding properties. KeywordsPeripheral cannabinoid receptor; CB2; Affinity tags; Chromatographic purification; Reconstitution; Functional; Heterologous expressionThe peripheral type cannabinoid receptor, CB2, belongs to the class A, rhodopsin-like G protein-coupled receptors (GPCR) 1 with a trans-membrane domain of seven helices. GPCRs are involved in a large array of physiological activities. While the human genome encodes several hundred different GPCRs, potentially making them one of the most important group of drug targets for pharmaceutical industry [1], the structure of only one of these receptors, bovine rhodopsin, is currently available at atomic resolution. Unlike rhodopsin, which is * Corresponding author. Fax: +1 301 594 0035. E-mail address: yeliseeva@mail.nih.gov (A. Yeliseev). NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript conveniently obtained from bovine rod outer segment discs in large quantities at high purity, the natural expression levels of most GPCRs in tissues are quite low, preventing their largescale purification as well as the application of most experimental methods to study their structure. In particular NMR and crystallography require heterologous expression of structurally unperturbed, functionally active GPCR. Heterologous expression of membrane proteins as a fusion with various expression partners in Escherichia coli cells is a commonly used technique that potentially yields milligram quantities of these polypeptides. N-terminal fusion with the maltose-binding protein (MBP) has been particularly beneficial for expression of recombinant membrane proteins in E. coli. It appears that the periplasmically localize...
Human cannabinoid receptor CB2 belongs to the class A of G protein-coupled receptor (GPCR). High resolution structural studies of CB2 require milligram quantities of purified, structurally intact protein. 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 CB2 with the resin, the double repeat of the Strep-tag was attached either to the N- or C-terminus of CB2 via a short linker. The CB2 was 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 demonstrate the high affinity of interaction between the Twin-Strep-tag fused to the CB2 and 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 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.
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