Recombinant Expression and Chromatographic Purification of Human Cannabinoid Receptor CB₂

Abstract

Human cannabinoid receptor CB₂ is an integral membrane protein that belongs to a large superfamily of seven transmembrane domain G protein-coupled receptors (GPCR). CB₂ is a part of the endocannabinoid system that regulates immune response, inflammation and pain sensitivity. The rational development of novel specific pharmaceuticals targeting this receptor relies on high resolution studies of the structure and mechanisms of activation of CB₂ . Here we review the methodology for recombinant expression and purification of CB₂ , suitable for preparation of multi-milligram quantities of functionally active receptor. CB₂ is expressed in a functional form in E. coli, solubilized in detergent micelles, and purified by tandem affinity chromatography. The recombinant CB2 is amenable to functional and structural studies by nuclear magnetic resonance spectroscopy and a wide range of biochemical and biophysical techniques.

Introduction

Heptahelical G protein-coupled receptors (GPCR) are integral membrane proteins involved in a wide array of cell signaling pathways. The cannabinoid receptor CB₂ that primarily expresses in cells of immune origin is an attractive target for the development of drugs for management of inflammation, immunological disorders and pain.^1-3 Structural studies can provide critical contribution to the rational design of novel specific drugs targeting this receptor.

The progress in structural studies of GPCR has been relatively slow, primarily due to the difficulties in obtaining large quantities of sufficiently pure, homogenous and functional receptors as well as conformational flexibility of GPCR that complicates preparation of well-diffracting crystals for X-ray crystallography. Recent improvements in expression techniques, methods of stabilization and crystal preparation resulted in over 30 high resolution structures of GPCR solved during the past few years, advancing our understanding of cell signaling.^4-6 In addition to X-ray crystallography, a large array of biophysical methods have been employed to study the structure and function of these receptors. In particular, applications of nuclear magnetic resonance (NMR) gain increasing attention since they allow studies of structurally unperturbed GPCR in native-like environments such as lipid bilayers.

With the exception of rhodopsin, most GPCR are present in native tissues at relatively low levels, and recombinant expression in a heterologous host is the only practical way to obtain these proteins in quantities required for structural studies. The commonly used expression systems include baculovirus-infected insect cells, yeast, bacterial or mammalian cells as well as cell-free systems.^7-12 However, the application of NMR to studies of GPCR require either site-specificor uniform labeling of these proteins with stable isotopes.¹² This can be achieved via metabolic labeling, through co-translational incorporation of labeled amino acids into the protein. The methods of stable isotope-labeling of proteins via expression in E. coli have been well developed,^13-15 while the use of insect- or mammalian cells for preparation of stable-isotope labeled proteins only recently became of practical interest, due to the high cost and complexity of the growth medium supplemented with labeled nutrients.¹⁶ The expression in yeast cells has also been used for production of several GPCR,¹⁷ but this host may not be suitable for preparation of some receptors because of the non-homogenous glycosylation or partial proteolysis of target proteins. Thus, the stable isotope labeling by expression in E. coli cells is currently the most cost-effective, practical alternative. Here we review the methodological approaches for expression and purification of CB₂ . These methodst are suitable for preparation of milligram quantities of functional GPCR for studies by NMR and a broad array of other biophysical techniques.

Functional Expression in E. coli

The general outline of the experimental strategy is given in Figure 1. Protein is expressed in E. coli membranes, and the expression levels and functional activity assessed by Western blot, ligand binding and G protein-activation assays.^_18-20 The purification is performed upon solubilization of the receptor in detergent micelles followed by the two rounds of affinity chromatography. The purified receptor can be studied either when solubilized in detergent micelles or after reconstitution into lipid bilayers in a form of proteoliposomes or nanodiscs.

Figure 1. Experimental strategy for preparation of functional CB₂

Multiple factors such as the choice of the bacterial host and expression vector, strength of the promoter, composition of culture media, temperature and length of the induction determine both the total yield as well as the fraction of correctly folded functional receptor. Several E. coli strains including BL21 (DE3), DH5a, KRX, Rosetta Gami, C41 and C43 were compared for their effectiveness in production of fusion CB₂ ; strain BL21 (DE3) provided the highest yield of the functional receptor.^{21, 22}

The expression partners fused to the target protein at the N-and C-termini can also dramatically affect the expression level and activity of the recombinant GPCR. A variety of plasmid constructs containing different combinations of expression- and solubility tags were tested for their efficiency in production of functional CB₂ . Figure 2 depicts selected expression constructs currently utilized in our laboratory.

We established that for proper folding and functionality of the recombinant CB₂ in E. coli, an appropriate N-terminal fusion partner is essential.¹⁹ For example, the N-terminal Haloalkane dehalogenase (Halotag) significantly increased the levels of the fusion CB2 protein. However, the accumulated receptor was not functional.²⁴ The expression of CB₂ without the N-terminal fusion partner resulted in only trace quantities of the recombinant protein.¹⁹ On the other hand, the N-terminal maltose-binding protein (MBP) was highly beneficial for the high-level functional expression of this receptor.²¹ The MBP is localized in the periplasm of E. coli; upon leaving the ribosome the nascent peptide is transported across the cytoplasmic membrane by the Sec translocon system.²⁵ This may facilitate the insertion of the downstream hydrophobic part of the fusion protein into cytoplasmic membranes. Importantly, the full length sequence of MBP is required for the maximal beneficial effect.¹⁹

The topology of the receptor in cytoplasmic membranes was determined by measuring the in vivo biotinylation of the fusion protein containing a biotin-recognition sequence placed in various positions relative to CB₂ . ²⁶ When expressed as a fusion with MBP, the N-terminus of CB₂ is exposed to the periplasmic space while its C-terminal part is localized to the cytoplasm of E. coli. While the MBP is believed to promote the insertion of the fusion protein into cytoplasmic membranes in an N-terminus-out orientation, the oxidative redox environment of the periplasm may facilitate the formation of the essential disulfide bond in extracellular loop,² thereby stabilizing CB₂ . The expression of the recombinant protein can be further increased by 30-40% by fusing the E. coli thioredoxin (Trx) to the C-terminus of CB₂ . Both MBP and Trx expression partners can be selectively removed if desired by cleaving the fusion with the specific TEV protease at recognition sequences flanking the receptor.

To enable the purification of CB₂ by tandem affinity chromatography, two small affinity tags can be added at the opposing ends of the recombinant protein. Examples of several such constructs are shown in Figure 2 and include His- and Strep-tag pair, His-tag/Bio-tag and Rho-tag/His-tag pairs, to name a few.

Figure 2. Schematic representation of constructs for expression of CB2 fusion protein in E. coli. (adapted from ^12,19,23)

The expression of CB₂ is performed from the low copy number vector based on the pMal-p2 backbone, from the weak lac promoter. The rather low rates of transcription allow better alignment of the protein synthesis with the rate of folding of the nascent polypeptide and its insertion into cytoplasmic membranes. For the same reason, lowering of the cultivation temperature from 37 °C to 20 °C has a clear beneficial effect for the functional expression of CB₂ , improving the yield of ligand binding-competent receptor from less than 20-30% to close to 100%.²² Please note though that further decrease of induction temperature to below 20 °C leads to significant drop in the protein expression levels and therefore is not practical.²²

For stable-isotope labeling of CB₂ a special protocol involving the expression in a minimal salt medium (MSM) supplemented with labeled nutrients was developed.^22,27 In order to compensate for the decrease of expression levels of the target protein in MSM, the cultivation of the E. coli cells in a fermenter was performed. Either glucose and ammonium salts or a mixture of amino acids can be used as sources of carbon and nitrogen. Under the controlled conditions of aeration, pH and nutrient supply the yield of the biomass in the fermenter can be increased 10-fold or higher, compared to the expression in rich 2xYT medium in shake-flasks. As much as 2-3 mg of the recombinant receptor can be obtained in 1L of fermentation culture.

Purification of the Recombinant CB₂

Purification of CB₂ is performed following its solubilization in detergent micelles. By screening solubilization conditions with ~ 40 different detergents, a mixture of the nonionic dodecyl maltoside (DDM) and zwitterionic CHAPS was identified as the most suitable for efficient extraction of the CB₂ from membranes (Table 1). Further supplementation with cholesteryl hemisuccinate (CHS) and a high affinity ligand results in significant stabilization of the receptor in detergent micelles.¹⁸

Table 1. Detergents for solubilization of recombinant CB₂.

Even when cultivated in a rich 2xYT medium, the E. coli cells accumulate CB₂ at moderate levels (~0.1% of total cellular protein) ⁽²¹⁾. The cultivation in a defined MSM medium results in even lower CB₂ content. Thus, a very efficient and selective chromatographic procedure is required to achieve the yield and purity of the target protein required for high resolution structural methods. Such chromatographic techniques as ion-exchange, size-exclusion or hydrophobic chromatography were not suitable, since the large detergent micelles prevented proper interaction of the receptor with the resin. On the other hand, high level purification and recovery of CB₂ was achieved by tandem affinity chromatography taking advantage of small affinity tags placed at opposing ends of the receptor.¹⁹

A typical purification protocol for fusion constructs of CB₂ containing C-terminal decahistidine tags begins with the IMAC chromatography. The binding of a shorter histidine tag (i.e. hexahistidine) to the Ni-NTA resin in the presence of detergent micelles was shown to be rather weak,²¹ while the longer decahistidine tag makes this purification step much more efficient and typically results in a recovery of ~80% or more of the fusion CB₂ . The fusion partners such as MBP can be removed after the elution from the resin, by cleaving the fusion protein with specific TEV protease at sites flanking the receptor.

The CB₂ released from the fusion is then re-captured via the N-terminal affinity tag (such as StrepTag), to separate it from TEV protease, cleavage products and other impurities. The capture of CB₂ on the affinity resin is performed in the presence of detergents DDM, CHAPS and CHS as well as salts and glycerol that stabilize the receptor. Since the affinity of a single Strep-tag (WSHPQFEK ) to the resin in the presence of detergent micelles is weak, a double repeat of this tag (twin-Strep-tag) is used to ensure a more efficient capture.19 The beneficial avidity effect was reported also for purification of soluble proteins on a StrepTactin resin.28 This double tag typically improves the recovery of the receptor to greater than 70-75%.

The expression of CB₂ in a defined minimal salt medium for stable isotope-labeling decreases the titer of the recombinant protein. To maximize the recovery of the target protein, the use of high affinity purification resin is highly desirable. The recently released StrepTactin XT resin satisfies this requirement, since it has an estimated affinity of interaction with the twin-Strep-tag in the low-nanomolar range (Figure 3).²⁹ The retention of the captured protein on a resin is very strong, which allows stringent washing conditions. The elution of the captured CB₂ is performed at mild conditions, upon addition of 50 mM biotin to the running buffer. This purification protocol allows recovery of up to 80% of the recombinant CB₂ originally present in the cell extracts, with high degree of purity (over 90%).

Figure 3. Surface plasmon resonance studies of binding of twin- Strep-tagged CB₂ to StrepTactin XT.

The recovered protein is highly pure and functionally active, as supported by the results of Ligand binding and G protein-activation experiments (Figure 4).

Figure 4. A, Ligand binding on CB₂ reconstituted into liposomes. B, activation of G protein on CB2 reconstituted into liposomes in the presence of agonist CP-55,940 (upper curve) or inverse agonist SR-144528 (lower curve).

To summarize, the protocols developed for recombinant production of CB₂ in E. coli cells and purification using tandem affinity chromatography yield the fully functional protein at high purity. Optimization of the expression plasmid, cultivation conditions and other parameters resulted in the recovery of the recombinant CB2 as high as 2-3 mg of protein from 1 liter of culture. The technology of preparation of the recombinant CB2 can be adapted to expression and purification of other GPCR and recombinant receptors. The developed methods for expression, stable isotope labeling, purification, stabilization and liposome reconstitution open a path to meaningful NMR spectroscopic studies on the CB₂ receptor.

Acknowledgements

This work was supported by the Intramural Research Program of the NIAAA, NIH.

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