Use of Tandem Affinity Chromatography for Purification of Cannabinoid Receptor CB₂

Abstract

Tandem affinity purification has been increasingly applied to isolation of recombinant proteins. It relies on two consecutive chromatographic steps that take advantage of the affinity tags placed at opposing ends of the target protein. This allows for efficient removal of contaminating proteins, including products of proteolytic degradation of the fusion that lack either N- or C-terminal tags. Here, we describe the use of two small affinity tags, a poly-histidine tag and a Strep-tag for expression and purification of the human cannabinoid receptor CB₂, an integral membrane G protein-coupled receptor.

1 Introduction

Affinity tags have become increasingly popular for purification of recombinant proteins.

We describe here the use of a combination of two small affinity tags, the polyhistidine tag [1] and a Strep-tag [2] for expression and purification of human cannabinoid receptor CB₂ [3], a class-A G protein-coupled receptor. The purification is performed in the presence of the nonionic detergent dodecyl maltoside (DDM), the zwitterionic detergent CHAPS as well as a derivative of cholesterol, cholesteryl hemisuccinate, required for stabilization of CB₂ in micelles. The necessity to perform purification in the presence of detergents constitutes a big challenge since they may affect the affinity and specificity of interaction between the tag and the resin by covering the tag and decreasing its accessibility [1].

The polyhistidine, or His-tag, usually contains 5–6 and, in some instances, as many as 10–14 consecutive histidine residues that bind to metal ions such as Ni⁺² or Co⁺² incorporated in different types of resins, allowing for purification of recombinant proteins via immobilized-metal affinity chromatography (IMAC) (Fig. 1a). The procedure described in this chapter is based on the Ni-NTA technology (Qiagen) [4]. The tetradentate chelant nitrilotriacetic acid occupies four valencies of the Ni⁺² ion, with two remaining valencies available for interaction with imidazole rings of histidine residues.

Fig. 1.

Ni-NTA affinity chromatography (a) and StrepTactin affinity chromatography (b). Chemical formulas of compounds required for chromatographic purification are shown

The binding step is usually performed at a pH of 7–8 to ensure that all histidine residues are deprotonated and, consequently, capable of interacting with nickel ions. The elution takes place in the presence of imidazole, which competes with histidine for the metal ions. While the high affinity binding of the His₆-tag to Ni-NTA resin has been reported (K_D = 14 nM [5]), both the affinity and specificity of binding can vary dramatically depending on the particular target protein, location of the tag, composition of the binding buffer, accessibility of the tag and the number of chelating residues. To improve the efficiency of interaction with the resin, the tag is usually placed at either N- or C-terminus of the target protein, where (in many cases) it is also less likely to interfere with the biological activity of the recombinant protein.

The second chromatographic step of our protocol involves purification on a StrepTactin resin using the repeat sequence of Strep-tag II (Fig. 1b). Strep-tag II is an 8-amino-acid polypeptide consisting of Trp-Ser-His-Pro-Gln-Phe-Glu-Lys that exhibits affinity (K_D for the binding is 1 µM) for an engineered streptavidin termed StrepTactin [6, 7]. Because of its small size, the Strep-tag usually does not interfere with the biological activity of its fusion partner. Furthermore, the purification is performed under mild conditions that preserve the bioactivity of most proteins. The elution of the purified recombinant protein from the resin by desthiobiotin, an analog of biotin, is specific since only Strep-tagged proteins are displaced from the interaction with the StrepTactin resin. The resin can be reused after regeneration with the HABA (hydroxy-azophenyl-benzoic acid, pH 8.0) which, when provided in excess, displaces desthiobiotin from the resin.

In this chapter we present protocols for expression of peripheral cannabinoid receptor CB₂ in E. coli, its extraction from membranes, the solubilization and stabilization of the recombinant receptor in detergent micelles, its affinity purification using His-tag followed by proteolytic removal of fusion partners by TEV protease, and its final purification via Strep-tag. To achieve efficient purification, a C-terminal His₁₀ as well as two identical Strep-tag sequences bridged by a 4-amino acid linker were attached to the N-terminus of CB₂ to increase the binding affinity to their respective resin [3]. Purification is performed under mild conditions to maintain the functional fold of the receptor. The purity of the resulting CB₂ can be determined by SDS-PAGE followed by Coomassie Blue staining or western blot, by assessing its biophysical properties in micelles through a variety of techniques including circular dichroism spectroscopy and NMR, or by evaluating its functionality by binding of radioligands and measuring its ability to activate cognate G proteins upon reconstitution into proteoliposomes (Fig. 2).

Fig. 2.

Flow chart of procedures for expression, purification and characterization of CB₂

2 Materials

2.1 Expression of CB₂ Fusion Protein and Preparation of Membranes

1. Plasmid pAY-125 for expression of CB₂.

2. BL21(DE3) competent cells.

3. Shaker-incubator with a temperature range capability between 20 and 37 °C.

4. 50 mg/mL Ampicillin stock solution: Dissolve ampicillin in water and filter the solution though a 0.22 µm filter.

5. 0.5 mM Isopropyl β-d-1-thiogalactopyranoside (IPTG) stock solution: Dissolve IPTG in water and filter through a 0.22 µm filter.

6. 20 % (w/v) Glucose stock solution: To 250 mL water slowly add 100 g of glucose while stirring, complete to 500 mL with water and sterilize by autoclaving.

7. Luria-Bertani broth (LB) medium: 10 g/L casein digest, 5 g/L yeast extract, 5 g/L NaCl. Dissolve powder in water and sterilize by autoclaving.

8. Baffled, 125-mL shake flasks. Sterilize by autoclaving.

9. Double-strength YT-medium (2 × YT): 16 g/L casein digest, 10 g/L yeast extract, 5 g/L NaCl. Autoclave 500 mL medium in 2 L baffled shake flasks.

10. Phosphate-buffered saline (PBS): 10 mM sodium phosphate, 150 mM NaCl, pH 7.4.

11. “Complete” protease inhibitor cocktail tablets (Roche).

12. Membrane storage solution: 10 mL of PBS adjusted to 20 % sucrose (2 g sucrose/10 mL PBS) with 1 tablet of “complete” protease inhibitor cocktail.

13. French press.

14. Handheld glass homogenizer.

2.2 Extraction, Solubilization and Stabilization of CB₂ in Detergent Micelles

1. Tris-buffered saline (TBS): 25 mM Tris–HCl, 130 mM NaCl, 2.7 mM KCl, pH 7.5.

2. EDTA-free protease inhibitor cocktail tablets (Roche).

3. DNAse I stock solution: 5 mg/mL DNAse I in water, approximately 1,000 U/mL.

4. 1 M MgCl₂ stock solution.

5. Anti foam A (Sigma).

6. 2× Solubilization buffer: 100 mM Tris–HCl, 400 mM NaCl, 60 % glycerol, pH 7.5.

7. 12× 3-[(Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS)/cholesteryl hemisuccinate (CHS) stock solution: 6 % CHAPS, 1.2 % CHS, store at 4 °C (see Note 1).

8. 10 % n-dodecyl-β-d-maltoside (DDM) stock solution, store at 4 °C.

9. 20 mM CP-55,940 stock solution: Dissolve CP-55,940 in ethanol. Store at −20 °C.

10. Cell homogenizer (Avestin) or a sonicator.

2.3 Purification

1. Ni-NTA agarose (Qiagen).

2. Buffer A: 50 mM Tris–HCl, 200 mM NaCl, 0.5 % [w/v] CHAPS, 0.1 % [w/v] CHS, 1 % [w/v] DDM, and 30 % glycerol, pH 7.5.

3. Buffer B: 50 mM Tris–HCl, 200 mM NaCl, 0.5 % [w/v] CHAPS, 0.1 % [w/v] CHS, 1 % [w/v] DDM, and 30 % glycerol, 250 mM imidazole, pH 7.5.

4. Buffer C: 50 mM Tris–HCl, 100 mM NaCl, 15 % glycerol, 0.5 % [w/v] CHAPS, 0.1 % [w/v] CHS, 1 % [w/v] DDM, pH 7.5.

5. TEV protease: Either commercially available or in-house-prepared recombinant tobacco etch virus (TEV) protease can be used. We express and purify the recombinant poly-His-poly-Arg-tagged protease according to established protocols [8 – 10]. Expression in BL21(DE3) harboring the expression plasmid and purification via Ni-NTA affinity chromatography followed by cation exchange chromatography are described in [11].

6. StrepTactin Poros column (EMD Biosciences).

7. 5 µM Desthiobiotin in Buffer A: Weigh the required amount of powder desthiobiotin (EMD Biosciences) in a small Eppendorf tube and carefully dissolve in 100–200 µL of 0.1 N NaOH. Mix with buffer A to achieve a final concentration of 5 µM (see Note 2).

8. Centrifugal spin concentrators with a 30–70 kDa molecular mass cutoff.

3 Methods

3.1 Expression of Fusion CB₂ in Shaker Flasks

The expression and purification protocols described here were developed for purification of the peripheral cannabinoid receptor CB₂, expressed as a fusion with several tags [3, 12] (see Note 3). The corresponding DNA construct, CB₂-125 is represented below (Fig. 3).

1. Inoculate 25 mL of LB medium supplemented with 25 µL of 50 mg/mL ampicillin in 125-mL baffled flask with E. coli BL21(DE3) cells harboring the expression plasmid (see Note 4).

2. Incubate overnight (ON) at 37 °C and 230 rpm agitation.

3. Prepare expression medium by adding 5 mL glucose solution and 500 µL ampicillin solution to the 500 mL of double-strength YT-medium flask immediately before inoculation. Use an appropriate number of flasks assuming the yield of ~400 µg of purified CB₂ protein from each flask.

4. Add 1–2 mL of the ON culture to each 2 L flask and incubate at 37 °C under agitation for 3–4 h until the optical density of the culture at 600 nm reached 0.4–0.5 U.

5. Lower the temperature of incubation to 20 °C (see Note 5).

6. Add 500 µL of IPTG solution to induce synthesis of the recombinant protein.

7. Add 62.5 µL of a solution of CP-55,940 ligand (20 mM in Ethanol, see Note 6).

8. Incubate for additional 38–40 h (see Note 5).

9. Collect cells by centrifugation at 4,000 × g for 30 min at 4 °C.

10. Resuspend and wash cells once in ice-cold PBS.

11. Collect cells by centrifugation as above and store pellet at −80 °C.

Fig. 3.

Fusion protein CB₂-125

3.2 Preparation of Membranes

We recommend testing the expression levels of the target protein in cell-membrane preparations obtained from small-scale cultures before proceeding with the large scale fermentation and purification. The following protocol is based on a previously published method for CB₂ expression [13]. Carry out all steps of membrane preparation on ice or at 4 °C.

1. Perform expression as described in Subheading 3.1, and collect cells from 25 to 50 mL of culture in a conical tube by centrifugation at 4,000 × g for 10 min. Alternatively, perform expression in a smaller scale format using 250 mL flasks and 50 mL of culture medium.

2. To 20 mL of cold PBS add 1 tablet of “complete” protease inhibitor cocktail.

3. Resuspended cells in a small volume (3–5 mL) of the cold PBS prepared in step 2.

4. Disrupt cells by passing the cell paste twice through a French Press.

5. Remove unbroken cells and cell debris by centrifugation at ~20,000 × g for 30 min at 4 °C.

6. Subject supernatant to a high-speed centrifugation at ~250,000 × g for 1 h at 4 °C.

7. Wash the resulting membrane pellet with cold PBS and resuspended in a small volume of membrane storage solution using a handheld glass homogenizer (see Note 7).

8. Flash-freeze small aliquots of the membrane suspension in liquid nitrogen and store at −80 °C until needed.

9. Proceed with evaluation of expression levels and functional activity of the target protein (see Note 8).

3.3 Extraction, Solubilization, and Stabilization of CB₂ from E. coli Cells

Starting from 10 L of cell culture (~80 g of wet biomass), typically 600 mL of crude extract can be obtained. Volumes of added solutions were calculated based on that number—adjust accordingly if necessary. Perform all the following procedures on ice or at 4 °C.

1. Transfer cell pellet (80 g) with a large spatula to a 1 L blender (see Note 9).

2. Add 100 mL of ice-could TBS buffer into the blender.

3. Add 2–3 tablets of EDTA-free protease inhibitor cocktail solubilized in 10 mL water.

4. Homogenize cell suspension in blender for 20–30 s.

5. Transfer the suspension on ice and start stirring.

6. Add 50 µL of DNAse I solution.

7. Add 3 mL of 1 M MgCl₂ to achieve a final concentration of 5 mM.

8. Add Anti-foam A until the foam disappears (20–40 µL).

9. Pass the suspension two times through a cell homogenizer (see Note 10).

10. Place the homogenized suspension in a 1,000-mL beaker on ice and start stirring.

11. Add 300 mL of 2× solubilization buffer (see Note 11).

12. Add 50 mL CHAPS/CHS 12× solution (see Note 11).

13. Add 60 mL of a 10 % DDM solution (see Note 11).

14. Add 300 µL of 20 mM ligand CP-55,940 solution (see Note 11).

15. Continue stirring for 40–60 min. Ensure that the final composition of the buffer in which CB₂ is solubilized is 50 mM Tris–HCl at pH 7.5, 200 mM NaCl, 0.5 % [w/v] CHAPS, 0.1 % [w/v] CHS, 1 % [w/v] DDM, 10 µM CP-55,940, 5 mM MgCl₂, and 30 % glycerol, supplemented with DNAse I (0.5 µg/mL) and EDTA-free protease-inhibitor cocktail.

16. Remove cell debris by centrifugation at a ~200,000 × g for 1 h at 4 °C.

17. Pass supernatant through a 0.45-µm filter and proceed with purification.

3.4 Ni-NTA Chromatography

All buffers used for purification should be supplemented with CP-55,940 at a final concentration of 10 µM. All procedures should be performed on ice or at 4 °C. Remember to take samples at each step to follow the purification process by SDS-PAGE and western blot (see Note 12).

1. Pack 8 mL of Ni-NTA resin to a suitable column compatible with an available chromatography work station (we use AKTA Purifier 100 [GE Healthcare]) allowing gradient-based chromatography and automated fraction collection.

2. Equilibrate the resin using at least 5 column volumes (CV) of buffer A + CP-55,940.

3. Load the filtered CB₂ solution at a flow rate of 0.5 µL/min. Maintain this flow rate for the entire purification.

4. Wash the resin with 20 CV of buffer A + CP-55,940 supplemented with 40 mM imidazole.

5. Elute protein with 10 CV of buffer B + CP-55,940 and collect 4 mL fractions (see Note 13).

6. Select the CB₂-containing fractions by SDS-PAGE followed by Coomassie Blue staining or by dot blot using anti CB₂ mAb or anti His-tag Ab (see Note 14).

3.5 Removal of Fusion Partners by Tobacco Etch Virus (TEV) Protease

1. Combine CB₂-containing fractions (typically 20 mL) and concentrate ~2.5-fold in centrifugal spin concentrators with a 30 kDa molecular mass cutoff (see Note 12).

2. Dialyze the concentrated sample against two exchanges of buffer C + CP-55,940 at 4 °C (see Note 12).

3. Add 0.5–1 mg of purified TEV protease to the dialyzed sample (5:1, CB₂: TEV, mol/mol) and allow digestion for at least 4 h or overnight at 4 °C (see Note 15).

4. Proceed with final step of purification or perform an intermediate purification step (see Note 16) to increase purity of the final product.

3.6 Purification via StrepTactin Affinity Tag

1. Pack a 3 mL-StrepTactin Poros column and operate it by gravity.

2. Equilibrate the resin with 5 CV of buffer C + CP-55,940.

3. Load the products of the TEV protease cleavage reaction (8 mL) onto the StrepTactin column.

4. Wash with 4 CV of buffer C + CP-55,940.

5. Elute CB₂ with 3 CV of buffer C + CP-55,940 supplemented with 5 µM desthiobiotin (see Note 17).

6. Combine elution fractions and concentrate in centrifugal spin concentrators with a 30 kDa molecular mass cutoff.

7. Dialyze against buffer A + CP-55,940 at 4 °C overnight.

8. Determine protein concentration with the Bio-Rad DC kit (see Note 18).

9. Aliquot the concentrated protein into Eppendorf tubes.

10. Freeze aliquots in liquid nitrogen and store at −80 °C until needed.

11. Evaluate purity of the preparation (see Note 19).

12. Characterize functional state of the purified protein (see Note 20).

13. The purified protein can be analyzed by a range of biophysical techniques (see Note 21).

Fig. 4.

Purification of CB₂-125. Coomassie Blue staining of a typical gel is presented. Lane 1, crude extract before 1st Ni-NTA chromatography; lane 2, concentrated fractions after 1st Ni-NTA chromatography; lane 3, fractions after dialysis against buffer B; lane 4, proteins after TEV protease digest; lane 5, combined fractions (flowthrough and wash) from the 2nd Ni-NTA column; lane 6, flowthrough from the StrepTactin column; lane 7, wash fraction from the StrepTactin column; lanes 8 and 9, eluate fractions from StrepTactin column, after concentration (5 and 2.5 µg of protein per lane, respectively)