Simplifying G Protein-Coupled Receptor Isolation with a Calcium

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Simplifying G protein-coupled receptor isolation with a calcium-dependent fragment complementation affinity system Niamh Ní Mhurchú, Lioudmila Zoubak, Gavin McGauran, Sara Linse, Alexei A Yeliseev, and David J. O'Connell Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00469 • Publication Date (Web): 29 Jun 2018 Downloaded from http://pubs.acs.org on June 30, 2018

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Biochemistry

Simplifying G protein-coupled receptor isolation with a calcium-dependent fragment complementation affinity system Niamh Ní Mhurchú,† Lioudmila Zoubak,‡ Gavin Mc Gauran,† Sara Linse,§ Alexei Yeliseev,*, ‡ and David J. O’Connell*,† † School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Ireland

‡ National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, 5625 Fishers Lane Bethesda, MD 20892, USA § Department of Biochemistry and Structural Biology, Lund University, Box 118, 221 00 LUND, Sweden *

To whom correspondence should be addressed: [email protected]; [email protected]

ABSTRACT The process of isolating recombinant G protein-coupled receptors from membrane preparations is challenging requiring solubilization in detergent micelles and multi-step affinity chromatography protocols. Solubilization buffers contain high concentrations of salts, detergents and glycerol that create stringent conditions necessary to stabilize the receptor but in which affinity chromatography resins perform poorly and these resins also require the addition of eluting agents that complicate downstream assays. To simplify this process we have developed a high affinity fragment complementation molecular switch as a highly specific system for receptor capture in solubilization buffer with a calcium chelation-based elution step releasing functional protein in a simple buffer. Here we describe in detail the design, methodology, interpretation, and limitations of this novel affinity chromatography system in the isolation and purification of the cannabinoid G protein-coupled receptor CB2, in comparison with commercially available systems. This powerful tool may be applied to any recombinant membrane bound protein and can be further optimized to enhance the yield and purity of the most challenging protein targets for study.

KEYWORDS. Affinity Chromatography, Purification, G protein-coupled receptor, Cannabinoid Receptor CB2, EF1-tag, Fragment Complementation

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INTRODUCTION G protein-coupled receptors (GPCRs) are one of the largest families of transmembrane proteins with 826 individual members identified in the GPCR Network and represent an important class of therapeutic drug target with 30–40% of all currently marketed drugs targeting these proteins (1,2). These 7-transmembrane proteins are particularly challenging to isolate in a pure form in quantities required for structural and functional studies, with a relatively small number of structures (15%) determined experimentally as a consequence (3). Isolation of GPCRs involves membrane solubilization in detergent-rich buffers and affinity chromatography capture using one or more tags fused to the N- or C-terminus of the protein of interest (4). However, commercially available affinity resins do not always provide the required yield or purity of target receptors in this buffer environment (5–7). We have previously achieved high level expression and recovery of the cannabinoid receptor CB2 (CB2) in Escherichia coli (E. coli) as a fusion with the N-terminal maltose-binding protein (MBP), and one or more affinity tags, including the polyhistidine tag (8), Streptavidin tag (9–11), and Rhodopsin tag (12). The relatively large MBP and TrxA sequences are intended to increase the solubility and yield and improve the correct folding of the recombinant receptor [8]. MBP could not be used for purification however, since the solubilization of the functional receptor from membranes required the use of maltoside-based detergents. The use of the high affinity purification tags, streptavidin tag and rhodopsin tag, involves elution of the recombinant receptor by displacing it from the resin with either biotin or a short amphiphilic peptide. These compounds readily partition into protein-detergent micelles and may interfere with some downstream applications. To simplify the process of receptor purification we have developed a molecular switch based on the highly specific, high-affinity fragment complementation between EF1 and EF2 domains of the Ca2+-binding protein calbindin D9k (13–17) as a powerful tool for receptor capture in solubilization buffer, with a calcium chelation-based elution step releasing functional protein in a simple buffer. This interaction is dependent on calcium ions and is governed by interactions between hydrophobic core residues in the alpha helices and loops of each domain (13–17), whereas charge separation between the two EF-hand members in a domain serves to diminish the electrostatic repulsion between the subdomains (13). This high-affinity complementation has previously been shown to favour assembly of GFP fragments and chromophore maturation in E. coli (18) providing an example of a calcium-dependent molecular switch in a complex environment (19). This highly specific complementation has the potential to provide a robust affinity purification system with chelation of calcium ions facilitating a gentle elution step in the complex buffer conditions required for functional GPCR isolation. Here we describe in detail the design, methodology, interpretation, and limitations of this novel affinity chromatography system in the isolation and purification of the cannabinoid G proteincoupled receptor CB2 in comparison with commercially available systems. MATERIALS & METHODS Chemicals and reagents. Synthetic genes were purchased from Integrated DNA Technologies. Restriction- and DNAmodifying enzymes were obtained from New England Biolabs. cOmplete™ His-tag resin was from Sigma-Aldrich and SulfoLink™ Coupling resin was from Thermo Scientific. Monoclonal


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Biochemistry

antibody NAA-1 against CB2 was from Eptomics Inc., anti-His antibody was from GE Healthcare. CP-55,940 was purchased from Tocris Cookson Inc. [3H]-CP-55,940 (specific activity 139.6 Ci mmol-1) and [35S]-γ-GTP (specific activity 1250 Ci mmol-1) were purchased from Perkin-Elmer. Inverse agonist of CB2 SR-144,528 was obtained from the National Institute on Drug Abuse (Research Triangle, NC). CM5 sensor chip, immobilisation reagents NHS, EDC, PDEA and ethanolamine, and HBS-EP buffers for SPR experiments were from GE Healthcare. Cholesteryl hemisuccinate Tris salt (CHS) and detergents 3-[(3-cholamidopropyl) dimethylammonio]-1propanesulfonate (CHAPS) and n-dodecyl-β-D-maltoside (DDM) were purchased from Anatrace. Lipids 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoylphosphatidylserine sodium salt (POPS) were purchased from Avanti Polar Lipids. DNA sequencing was performed by MWG Eurofins. In all cases, E. coli BL21 (DE3) cells (Agilent Technologies) were used for cloning. Protein analysis. Recombinant CB2 fusion proteins were detected by Western blot with rabbit monoclonal NAA-1 antibody raised against CB2 and mouse monoclonal anti-His6 antibody (Qiagen). Western blot was developed with anti-rabbit HRP antibody (1:5,000) or anti-mouse HRP antibody (1:5,000) and visualised by chemiluminescence by Gel Logic imaging system (Kodak). Protein concentration in the presence of detergents was determined using the DC™ Protein Assay Reagent (BioRad), with BSA as protein standard, and/or UV-Vis spectrophotometry (Agilent Technologies). The purified proteins were concentrated to 0.5–2.0 mg/mL on centrifugal filter devices (Amicon) and stored at −80oC. TEV protease cleavage of CB2 fusion protein TEV protease with a C-terminal His-tag was expressed in E. coli BL21-RIL harbouring plasmid pRK793 as described previously [8]. The purified fusion proteins were dialyzed against buffer C with reduced concentrations of glycerol and NaCl (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 10% glycerol, 0.5% CHAPS, 0.1% CHS, 1% DM), supplemented with 10 µM CP-55,940 for 1 h at 4 °C, and digested (0.1 mg protease per 0.5 mg fusion protein) for 4 hours or overnight. Surface plasmon resonance. All surface plasmon resonance (SPR) experiments were performed with a BiaCore™ T200 SPR instrument (GE Healthcare).


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RESULTS Overview of the calcium-dependent fragment complementation affinity system. The novel affinity purification system described herein successfully achieves highly specific, high affinity immobilisation of a set of EF1 tagged CB2 GPCR proteins. This system relies on the binding of calcium ions by the EF hand fold in the EF1 affinity tag peptide and in the EF2 affinity ligand coupled to the agarose resin. When calcium is coordinated by the amino acid residues in the EF hands the helix-loop-helix secondary structure of the protein domains is restored and this facilitates high affinity fragment complementation between them, mediated by interactions between hydrophobic core residues in the alpha helices and separation of charges, creating a calcium dependent molecular switch facilitating a mild elution step involving the chelation of calcium (Figure 1).

Figure 1. Schematic representation of an EF1 tagged CB2 GPCR with EF2 agarose. In the absence of calcium the peptide domains lack the helix-loop-helix secondary structure. When calcium is bound the domains are correctly folded and mediate highly-specific, high-affinity fragment complementation that is the basis for the affinity purification. EF1 affinity-tagged CB2 constructs and EF2 agarose affinity resin. 1. Bovine minor A calbindin D9K EF1 sequence was optimised for heterologous recombinant expression in E. coli using a codon frequency table and manual selection of the most abundant codon for each amino acid. The codon-optimised gene for the EF1-affinity tag (KSPEELKGIFEKYAAKEGDPNQLSKEELKLLLQTEFPSLLKGP), in combination with one or more


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Biochemistry

fusion-epitope sequences, was synthesised by Integrated DNA technologies, Inc for insertion into pMAL-p2 plasmid derivitives developed in our previous work. 2. Plasmid CB2-130 (20) was digested with PlutI/BamHI (CB2-351) or BsaAI/BamHI (CB2-352) to excise the Twin Strep-II affinity tag sequence. Plasmid CB2-260 was digested with NotI/HindIII (CB2-353, CB2-354) to remove the biotin–protein ligase recognition site (BRS) sequence. 3. Excised sites were replaced with the recombinant synthetic gene inserts described above. 4. Recombinant CB2 fusions in E. coli BL21 (DE3) cells were expressed in double strength YT broth (16 g digest peptone, 10 g yeast extract, and 5 g NaCl per litre), supplemented with ampicillin (50 mg L-1) and glucose (0.2% (w/v)) as described previously (21). 5. E. coli membranes expressing EF1-tagged CB2 were prepared for western blot analysis per the previously published protocol (8). 6. Fusion proteins from six expression constructs (Figure 2 A) were confirmed to express in the membrane with anti-CB2 (Figure 2 B (i)) and anti-His (Figure 2 B 9ii)) antibodies. 7. EF2 agarose was synthesised by thiol coupling of reduced EF2-GGC peptide (STLDELFEELDKNGDGEVSFEEFQVLVKKISQGGC) to SulfoLink™ Coupling Resin (ThermoFisher). 8. 1 mg/mL of EF2-GGC was reduced in coupling buffer (50mM Tris, 5mM EDTA-Na; pH 8.5) plus 1 mM DTT for 1 hour at room temperature before removal of DTT by size exclusion with a NAP10 column. 9. The reduced peptide was then incubated with 2 mL of a 50% (w/v) slurry of SulfoLink™ resin equilibrated in coupling buffer for 15 minutes with end over end mixing followed by 30 minutes stationary incubation. 10. The peptide solution was drained from the slurry and the resin washed with three resin bed volumes of coupling buffer before blocking in 2 mL of 50 mM L-Cysteine.HCl in coupling buffer using the same reaction times used for peptide coupling. 11. The resin was drained and washed with 6 resin bed volumes of 1 M NaCl prior and was washed three times in 10 mM HEPES, 150 mM NaCl, 2 mM CaCl2 prior to use.

Figure 2. Qualitative analysis of expression of EF1-CB2 protein fusions in E. coli membranes. (A) Expression constructs of EF1 tagged CB2 fusion proteins (352, 353, 354) with parent CB2 constructs (130, 125, 260). (B) Detection of recombinant fusion CB2 proteins in membrane


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preparations from E. coli after separation on SDS PAGE using (i) anti-CB2 monoclonal antibody and (ii) anti-His tag monoclonal antibody. Full length fusions indicated by asterisks. Single step EF1-CB2 capture with EF2 agarose. Purification protocols for EF1 tagged constructs CB2-352, CB2-353 and CB2-354 expressed in E. coli membranes were devised using an EF2 peptide ligand functionalised agarose resin. 1. The addition of amino acids Gly-Gly-Cys at the C-terminus of the 35-amino acid peptide facilitated thiol coupling of approximately 0.5 mg EF2-GGC/mL (w/v) agarose resin. The ability of this resin to capture EF1-tagged proteins in the glycerol and detergent rich solubilization buffer was first tested using a soluble enzyme, carbonic anhydrase, with an N terminal EF1 tag (Figure S1). Successful capture and elution of this cytosolic protein from E.coli expression culture confirmed the potential of a single step protocol for membrane bound receptor purification using this resin. The binding capacity of this resin for expressed EF1-CB2 fusions in solubilisation buffer was measured using a single step protocol for capture of the EF1 tag either at the amino terminus of CB2, centrally located within the CB2-352 fusion protein, or at the Cterminus of CB2-353 and CB2-354 fusions. 2. CB2 fusion proteins from E. coli cells were extracted and clarified in solubilisation buffer (50 mM Tris-HCl pH 7.5, 200 mM NaCl, 30% glycerol, 0.5% CHAPS, 0.1% CHS, 1% DM), supplemented with 10 µM CP-55,940 and 4 mM CaCl2. 3. 50, 100 and 150 ml of clarified lysate was then incubated overnight with 1 mL of EF2 agarose resin at 4°C on rotary shaker and collected the following day in a disposable column. 4. The resin was washed with 20 column bed volumes (CV) of solubilisation buffer containing 4 mM CaCl2. 5. Fusion CB2 protein was eluted in 6 fractions each of 1 resin bed volume of 50 mM EDTA in solubilisation buffer. 6. The eluted fractions 1–6 from each purification (50, 100 and 150 mL) were then pooled and concentrated using Amicon Ultra 15 cellulose filters with a 3 kDa cut off. 7. Electrophoresis of concentrated, captured proteins eluted from the resin, and subsequent dye staining and immunoblotting confirmed specific capture of the fusions irrespective of tag location (Figure 3). The 92 kDa full length MBP-CB2-352 fusion and a 47 kDa CB2 protein released by treatment with TEV protease were both efficiently recovered from dilute preparations, as confirmed by anti-CB2 and anti-His Western blot (Figure 3A (i) and B (i)). The auto-cleavage of the MBP-CB2 fusion protein prior to or during the purification procedure at the N-terminally located TEV cleavage site is significant, although the strong signal seen on the stained gel is due to both the 47 kDa fragment and the 45 kDa MBP-containing fragment and the associated signal for the CB2 fragment is lower than that for the full-length protein indicating that some of the fusion protein is still uncleaved. Partial self-cleavage of the MBP-CB2 fusion constructs prior to TEV protease treatment has been observed for CB2-125 and CB2-130 constructs previously (20,21). The 106 kDa CB2-354 receptor fusion is efficiently enriched to a high level of purity in this single step despite evidence of limited proteolysis evident from the immunostaining at multiple molecular weights. It is important to take into account though that the intensity of bands in the Instant Blue-stained gels do not adequately represent the true purity of the CB2 preparations, since the bulk of hydrophobic CB2 protein is screened from interaction with the dye by a large detergent belt. Therefore, the intensity of the band corresponding to the purified CB2 protein is significantly weaker than that of the soluble E.coli proteins and the amount of CB2 on a gel is significantly underrepresented by conventional staining. Therefore, the Western blot probed with anti-His-tag antibody provides a more


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Biochemistry

adequate assessment of the relative content of the His-tagged CB2. While the majority of the protein is uncleaved there is a consistently strong signal for a 61 kDa band seen with both antiCB2 and anti-His Western blots (Figure 3A (ii) and B (ii)). The highest yield of purified receptor fusion from 5 L expression cultures was recorded for recombinant CB2-353 and CB2-354 (0.83 mg and 1.16 mg, respectively), whereas the yield of recombinant CB2-352 fusion was lowest (0.66 mg) (Table 1). These are satisfying yields of protein when compared with a commercially developed affinity resin, StrepTactin XT (IBA), which delivered 1.5 mg of purified CB2-130 fusion protein in similar expression/ purification protocol (Table 1). C-terminal EF1 tag location in combination with solubility enhancing sequences (TrxA) appears to be optimal for purification although it is potentially useful that a central location of the EF1 tag can still interact effectively with immobilised EF2.

Figure 3. Single step capture of EF1-CB2 protein fusions on EF2-GGC agarose. (A) Schematic of the CB2-352 fusion construct with a centrally located EF1 tag highlighted in green (i) and CB2354 fusion construct with a C terminal EF1 tag highlighted in green (ii). Immunoreactive polypeptides are indicated with solid bars above and beneath. (B) Detection after SDS PAGE separation of purification products with Instant Blue gel staining and immunoblotting: (i) CB2352 and (ii) CB2-354. 1–3, increasing input volumes of 50, 100 and 150 mL; 4–6, corresponding flow through fractions; 7–9, pooled elution fractions from a total of 6 individual fractions of one bed volume each. Full length fusions indicated by asterisks.

Fusion construct

Molecular weight (Da)

CB2-130

89016.3

Yield of CB2 fusion protein (mg protein) 1.5


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CB2-352

92015.8

0.66

CB2-353

92056.9

0.83

CB2-354

106001.6

1.16

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Table 1. Quantitative analysis of EF2 Agarose in Single Step Chromatography. Comparisons are based on yield of CB2 fusions from 5 litres of expression culture with 1 mL of affinity resin. CB2130 control fusion was purified on commercially available StrepTactin XT resin using manufacturer’s instructions with modifications previously published. Tandem affinity purification of EF1-CB2 recovery with EF2 agarose and IMAC. 1. CB2-352, 353 and 354 fusion proteins first isolated with EF2 agarose were then dialyzed against buffer C (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 10% glycerol, 0.5% CHAPS, 0.1% CHS, 1% DM), supplemented with 10 µM CP-55,940 for 1 h at 4 °C, and treated with TEV protease (0.1 mg protease per 0.5 mg fusion protein) for 4 hours or overnight prior to a second affinity purification of the decahistidine tag on Ni-NTA agarose resin. 2. cOmplete™ His-tag resin was added to the EF2 purified fusion proteins and the captured Histagged CB2 protein was eluted with 250 mM imidazole in 50 mM Tris-HCl pH 7.5, 200 mM NaCl, 30% glycerol, 0.5% CHAPS, 0.1% CHS, 1% DM. Fully cleaved constructs have expected molecular weights of 47 kDa for CB2-352 and CB2-353 and 43 kDa for CB2-354 (Figure 4A). 3. Electrophoresis of tandem affinity purified fusions (T) was followed by dye staining (InstantBlue) and identified successful purification of each of the predicted molecular weight fusions (Figure 4B). It is evident that the TEV recognition site at the N terminus of CB2 was cleaved efficiently in all three constructs, but it is also clear from this data that the second TEV cleavage site at the C-terminus of CB2 in CB2-354 was not fully cleaved in the reaction leading to the isolation of two CB2 fusion fragments of 61 kDa (partial digest) and 43 kDa (full digest). Again the limitations of dye staining here should be considered in visualising the purified CB2 monomer and also in showing the removal of the soluble E.coli proteins from the preparation.

Figure 4. Tandem affinity purification of EF1-CB2 fusion proteins with EF2 agarose and IMAC. (A) Schematic representation of proteolytically cleaved EF1-CB2 fusion constructs. (B) Detection after SDS PAGE separation of affinity purified CB2 proteins with Instant Blue gel staining: (S) single step purification on EF2 agarose and (T) tandem affinity purification on EF2 agarose


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Biochemistry

followed by TEV cleavage and IMAC purification on Ni-NTA agarose. Fully digested CB2 fusions indicated by asterisks. Measurement of the binding affinity of the calcium-dependent fragment complementation affinity system in solubilization buffer with surface plasmon resonance. 1. All surface plasmon resonance (SPR) experiments were performed with a BiaCore™ T200 SPR instrument (GE Healthcare). 2. Immobilization of EF2-GGC to the sensor chip was performed through ligand thiol disulphide exchange coupling as follows. Briefly, 10 mM HEPES/NaOH at pH 7.4, 0.15 M NaCl, 0.005% TWEEN® 20, and 3.4 mM EDTA was used as flow buffer during coupling at a constant flow rate of 5 µL/min. 3. Equal volumes of 0.1 M NHS and 0.4 M EDC were first mixed, and 25 µL of the mixture were allowed to flow over the series S CM5 sensor-chip surface to activate the carboxymethylated dextran (5 min). 4. Twenty microliters of a PDEA solution, made by dissolving 4.5 mg PDEA in 205 µL 0.1M borate buffer at pH 8.5, were injected to introduce a reactive disulphide group onto carboxyl groups of the sensor chip. 5. EF2-GGC at 0.1 mg/mL in 70 µL 10 mM sodium formate buffer at pH 4.3 was then injected over the sensor chip. The C-terminal Cys of EF2-GGC was used to create a covalent link between the immobilized EF2-GGC and the dextran matrix of the sensor chip leading to immobilisation of 250 RU of EF2-GGC. 6. Deactivation of the excess reactive disulphides on the chip was done by injecting 20 µL of 50 mM L-cysteine with 1 M NaCl in 100 mM formate buffer at pH 4.3. At least one of the four flow cells on each sensor chip was reserved for a blank immobilization with no protein in the coupling step and was used as reference. 7. The kinetics of binding measurements between EF1-CB2 fusion proteins and immobilised EF2 were performed at 10°C in solubilization buffer supplemented with 2 mM CaCl2 at flow rates of 10 and 30 µL/min. Experiments were conducted at 10oC due to the known instability of CB2 in detergent micelles at higher temperatures (11,22) 8. After each experimental cycle, the surfaces were regenerated by injecting 10 mM EDTA, pH 8, for 2 min to remove residual-associated fusions. 9. Data analysis was performed using BIAevaluation software (BiaCore™) and KaleidaGraph software (Synergy Software) with the following equations for dissociation phase Y = B + A exp(−koff t) and association phase Y = B + A(1 − exp(−(C kon + koff)t))C kon / (C kon + koff)where A is a fitted constant, B is a constant baseline, C is the injected protein concentration, and kon and koff are the fitted association and dissociation rate constant, respectively. The results of the SPR experiments confirming high affinity fragment complementation between EF1-CB2 and EF2 affinity ligand in membrane solubilization buffer are shown in Figure 5. The relative response for EF1 tagged CB2352 and CB2-354 at 100 nM was measured and compared with the EF1 peptide at 100 nM. The maximum response of CB2-352 and CB2-354 were very similar at 188 and 184 RU respectively


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(Rmax 298 and 276 RU) (EF1 = 74 RU/ Rmax 277 RU), and the dissociation rate constant, koff, was estimated by fitting a single exponential decay plus constant baseline to the dissociation phase data at a single concentration of 100 nM (Figure 5A). The value obtained for CB2-352 (koff = 8 x 10-5 s-1) (SD = 1 x 10-6 s-1) was marginally faster than that for CB2-354 (koff = 5 x 10-5 s-1) (SD = 1 x 10-7 s-1) and the sensorgrams are seen to cross early in the dissociation phase despite equivalent binding in the association phase. An equivalent dissociation rate constant to CB2-354 was measured for the control EF1 peptide alone (koff = 5 x 10-5 s-1) (SD = 1 x 10-7 s-1) based on data recorded using the same sensor chip (Figure 5A). The association rate constant was estimated by fitting to the association phase data (assuming fixed koff as obtained above) for a concentration series (12.5 – 100 nM) of CB2-354 in solubilization buffer as kon = 2 x 104 M-1 s-1 (SD = 1.9 x 102 M-1 s-1) (Figure 5 B) and CB2-352 kon = 2 x 104 M-1 s-1 (SD = 3.5 x 102 M-1 s-1) (Figure S2). A calculated KD value was obtained in each case as the ratio KD = koff/kon with each fit to the association phase at each concentration shown and a single fit to the dissociation at 100 nM also shown (Figure 5B and S2).

FIGURE 5. SPR analysis of the interaction between EF1-CB2 fusion proteins and EF2-GGC functionalised surface in solubilization buffer. (A) Sensorgrams showing the relative response recorded during injection of CB2-352 (black), CB2-354 (red) and EF1 peptide (yellow) at a concentration of 100 nM, followed by buffer flow, over the EF2-GGC surface. (B) Fitting to (i) the


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Biochemistry

association phase in a concentration series of 100 nM (red), 50 nM (orange), 25 nM (green) and 12.5 nM (blue) CB2-354, and to (ii) the dissociation phase after injecting 100 nM CB2-354. The equilibrium dissociation constant, KD, for the CB2-354 EF2 interaction was estimated from the ratio of the fitted rate constants as KD = koff / kon = 3.10-9 M and 4.10-9 M for CB2-352. Activity assay of purified receptor reconstituted in liposomes Reconstitution of the purified CB2 protein into liposomes was performed using Pierce detergent removal resin (Thermo Scientific). 1. Purified protein (13 µg) in solubilisation buffer was mixed with 132 µg of lipids POPC/POPS (4/1, mol/mol) dissolved at a concentration of 3.3 mg/mL in 1% (w/v) CHAPS (22). 2. The solution (80 µL) was loaded onto a 0.5 mL Pierce detergent removing spin column and resulting proteoliposomes were eluted following manufacturer’s instructions. 3. The obtained proteoliposomes were identified as unilamellar, with a mean diameter of ~150 nm, using dynamic light scattering (Beckman Coulter model N4 Plus) [25] and Nanosight measurements. 4. Myristoylated Gαi1 was expressed in E. coli and purified as previously described (23). 5. Recombinant human β1γ2 subunits of G protein were expressed in baculovirus-infected Sf9 cells and purified as described earlier (24). The activity assay was conducted as follows (final concentrations in 50 µL reaction mixture are given in parentheses): 6. The membrane or liposome sample was diluted into ice-cold 10 mM MOPS buffer, pH 7.5, to a receptor concentration of 2 g/L or 0.2–0.8 mg/L, respectively. 7. Ten microlitres of the diluted dispersion containing the receptor were dispensed into presiliconised glass tubes and mixed with 2 µM CP-55,940 in MOPS buffer containing 0.1% (w/v) BSA. 8. After addition of a mixture of Gαi1 (100 nM) and Gβ1γ2 (500 nM), the tubes were incubated on ice for 30 min. 9. The reaction was initiated by addition of 50 mM MOPS buffer pH 7.5, supplemented with 1 mM EDTA, 3 mM MgCl2, 4 µM GDP, 0.3% (w/v) BSA, 100 mM NaCl, 1 mM DTT, and 35S-γ-GTP (2– 6 nM, 1250 Ci mmol-1), followed by rapid transfer of the tubes to a water bath at 30°C. The incubation continued for 20 min. 10. The reaction was terminated by addition of 2 mL of ice-cold stop solution, TNMg buffer (20 mM Tris-HCl pH 8.0, 100 mM NaCl, and 25 mM MgCl2) and rapidly filtered through nitrocellulose filters (Millipore, Billerica, MA). 11. Each filter was washed four times with 2 mL of cold TNMg buffer, dried, placed in scintillation vials filled with ScintiSafe Econo F scintillation liquid (Fisher, Waltham, MA), and the radioactivity counted. 12. Functional activity of purified CB2 fusions reconstituted in POPC/POPS lipid bilayers was then measured by binding of [35S]-GTPγS to activated cognate heterotrimeric G protein (Figure 6). The assay was performed on CB2-352 and CB2-354 purified by tandem affinity chromatography on EF2 and Ni-NTA resins, and the control construct CB2-130 (IMAC and Streptactin XTpurified). All purified fusions tested demonstrated high levels of G protein activation, with CB2352 showing the highest level of activity represented as 100% activity for relative comparison (Figure 6, Table S1). Despite the seemingly lower activation level of CB2-354 (78%) when compared to CB2-352 and assay control CB2-130 (85%), the difference is not significant and


Biochemistry

indicates that the position of the relatively small EF1-tag at the N- or C-terminal end of CB2 does not adversely affect the function of the receptor.

100

Relative activity per ng CB2 (%)

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80 60 40 20 0

FIGURE 6. Quantitative assay of functional activity of purified CB2 protein in liposomes. Result of G protein activation assay showing the percent activity of CB2 fusions. The sample with the highest levels of activation was used as an activity standard (set to 100%). Limitations The data presented in this study demonstrates for the first time that calcium-dependent fragment complementation is effective for purification of functional integral membrane receptors with high yield. Specifically, we report the use of calcium-dependent high affinity binding between the EF hand sub-domains of bovine calbindin D9k, with the structure encoded specificity dictated by the side chains of the hydrophobic core residues in the alpha helices and loops of these fragments. However, the single step protocol based on EF-hand complementation did not deliver pure monomeric fusion protein with a significant number of immunoreactive bands at multiple molecular weights seen. This may be due to proteolytic cleavage of the fusions during expression, with possible multimerization of fusion construct protein fragments during purification. Simple improvements to the protocols used such as optimizing the incubation time with the affinity resin used in the purification protocol on a timescale that limits proteolytic degradation should lead to superior single step results. CB2 protein, similar to many other GPCR, tends to form dimers and higher order oligomers in the presence of SDS, which result in multiple high-molecular weight bands in SDS-PAGE in addition to the “true monomer” of CB2. This further complicates the assessment of the purity of the receptor by gel electrophoresis. However, the results of the ligand-binding assay performed on CB2 purified with tandem affinity protocol indicate that the protein preparation was at least 90% pure since it was capable of binding specific cannabinoid ligand assuming 1:1 stoichiometry of the protein-ligand complex. Improving the yield of purified receptor fusions is also desirable and this may be achieved through optimisation of the coupling efficiency of the EF2 affinity ligand to the agarose resin to


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Biochemistry

further enhance the capacity for EF1-CB2 fusion proteins. The twin TEV cleavage site approach was not entirely successful with a failure of cleavage at the C terminal TEV cleavage site possibly due to steric hindrance of access of the protease to this cleavage site. The sequence of amino acids adjacent to the TEV recognition sequence at the C-terminus of CB2 and proximal to the solubility enhancing TrxA sequence may be further optimised to enable a complete digestion of this fusion protein. The tandem purification of CB2-352 and CB2-353 was successful in purifying the fully cleaved protein but the Ni-NTA resin in the second purification step also co-purified the lower molecular weight 6xHis-tagged TEV protease (27 kDa) (Figure 4B). The amount of protease used in this reaction can be titrated to a lower minimum concentration as the Nterminal cleavage site is already subject to some autocleavage as discussed earlier and the protease can be removed from the preparation post cleavage with a further chromatographic step such as size exclusion chromatography. Such contamination may be avoided completely with the use of a non-His-tagged protease. The greater purity of constructs CB2-353 and CB2-354 after tandem affinity purification reflects the superior performance of the initial EF2 agarose purification step that is likely due to the greater accessibility of the EF1 tag at the C terminus in the fusion proteins. Our data demonstrate that these expressed receptors are functional receptor proteins, bind specific ligands and activate G proteins in proteoliposome preparations of purified receptors. The high affinity of binding associated with fragment complementation between EF1 and EF2 peptides is maintained in SPR measurements while the low temperature, high detergent and glycerol content in solubilization buffer was seen to lower the rate of association previously recorded between EF1 and EF2 peptides in non-solubilization buffer. The slow dissociation rate however is sufficient for high affinity capture and complete dissociation upon calcium chelation was maintained. In summary, our data demonstrate that using the EF1 affinity tag as a fusion partner for CB2 GPCR expression and purification is a promising strategy for achieving single step recovery of functional receptors, and that the mild elution associated with this molecular switch is a potentially significant advantage for numerous downstream applications. Furthermore, optimization of this method with enhanced capacity affinity resin to enable reduction of the number of purification steps and processing time will likely limit nonspecific proteolysis of the recombinant receptor and increase the recovery of the intact target protein. In the future, this strategy could be utilized to deliver pure, functional membrane proteins in a simple buffer preparation. Acknowledgements We wish to gratefully acknowledge the funding support of Science Foundation Ireland in making this collaborative study possible, specifically grant numers: 12/IP/1620, 13/TIDA/B2679 and 14/TIDA/2427. Supporting Information Figure S1. EF2 agarose purification of EF1-Carbonic anhydrase in solubilization buffer. Figure S2. SPR analysis of the interaction between CB2-352 fusion and EF2-GGC functionalised surface in solubilization buffer. Table S1. Raw data of G protein activation at the CB2 proteoliposome measured in counts per minute (CPM).


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