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Application of Strep-Tactin XT for affinity purification of Twin-Strep-tagged CB2, a G protein-coupled cannabinoid receptor.

ABSTRACT: Human cannabinoid receptor CB2 belongs to the class A of G protein-coupled receptor (GPCR). CB2 is 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 CB2 require milligram quantities of purified, structurally intact protein. While we previously reported on the methodology for expression of the recombinant CB2 and 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 CB2 with 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 CB2 via 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 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 on the purified protein 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 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.

SUBMITTER: Yeliseev A

PROVIDER: S-EPMC5406253 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Application of Strep-Tactin XT for affinity purification of Twin-Strep-tagged CB2, a G protein-coupled cannabinoid receptor.

Yeliseev Alexei A Zoubak Lioudmila L Schmidt Thomas G M TGM

Protein expression and purification 20161117

Human cannabinoid receptor CB2 belongs to the class A of G protein-coupled receptor (GPCR). CB2 is 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 CB2 require milligram quantities of purified, structurally intact protein. While we previously reported on the methodology for expression of the recom ...[more]

PMID: 27867058

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Project description:The endocannabinoid system (ECS) consists particularly of cannabinoid receptors 1 and 2 (CB1 and CB2), their endogenous ligands, and enzymes that synthesize and degrade their ligands. It acts in a variety of organs and disease states ranging from cancer progression over neuropathic pain to neurodegeneration. Protein components engaged in the signaling, trafficking, and homeostasis machinery of the G-protein coupled CB2, are however largely unknown. It is therefore important to identify further interaction partners to better understand CB2 receptor functions in physiology and pathophysiology. For this purpose, we used an affinity purification and mass spectrometry-based proteomics approach of Strep-HA-CB2 receptor in HEK293 cells. After subtraction of background interactions and protein frequency library assessment we could identify 83 proteins that were classified by the identification of minimally 2 unique peptides as highly probable interactors. A functional protein association network analysis obtained an interaction network with a significant enrichment of proteins functionally involved in protein metabolic process, in endoplasmic reticulum, response to stress but also in lipid metabolism and membrane organization. The network especially contains proteins involved in biosynthesis and trafficking like calnexin, Sec61A, tubulin chains TUBA1C and TUBB2B, TMED2, and TMED10. Six proteins that were only expressed in stable CB2 expressing cells were DHC24, DHRS7, GGT7, HECD3, KIAA2013, and PLS1. To exemplify the validity of our approach, we chose a candidate having a relatively low number of edges in the network to increase the likelihood of a direct protein interaction with CB2 and focused on the scaffold/phagosomal protein p62/SQSTM1. Indeed, we independently confirmed the interaction by co-immunoprecipitation and immunocytochemical colocalization studies. 3D reconstruction of confocal images furthermore showed CB2 localization in close proximity to p62 positive vesicles at the cell membrane. In summary, we provide a comprehensive repository of the CB2 interactome in HEK293 cells identified by a systematic unbiased approach, which can be used in future experiments to decipher the signaling and trafficking complex of this cannabinoid receptor. Future studies will have to analyze the exact mechanism of the p62-CB2 interaction as well as its putative role in disease pathophysiology.

Dataset Information

Application of Strep-Tactin XT for affinity purification of Twin-Strep-tagged CB2, a G protein-coupled cannabinoid receptor.

Publications

Application of Strep-Tactin XT for affinity purification of Twin-Strep-tagged CB<sub>2</sub>, a G protein-coupled cannabinoid receptor.

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