Emulsiflex c3

To prepare the base system, a high-water content (W/O of 40/60, v/v), and an emulsifier content of 6% (total emulsion-basis, % (v/v)), were chosen. Three emulsifier systems were tested, namely S80:T80 54:46 (v/v), S80:T80 80:20 (v/v), and S85:T80 80:20 (v/v). The chosen compositions and contents were based on previous published works [7 (link),22 (link),23 (link)]. The preparation method, developed in-house, comprised, firstly, the preparation of a primary emulsion, followed by the application of successive HPH cycles (12, 21, and 24). The primary emulsion comprised the addition of the emulsifier mixture to the water phase for homogenization (10 min under stirring) followed by the addition of the oil phase and homogenization using a Unidrive X1000 Homogenizer Drive (CAT Scientific, Germany) at 11,000 rpm for 5 min. An aliquot of this primary emulsion was withdrawn for microscopic analysis. Afterwards, the primary emulsion was subjected to successive HPH cycles using an EmulsiFlex-C3 (Avestin, Canada) at 1500 bar.
To prepare the emulsions loaded with the cinnamon (Cinnamomum zeylanicum) aqueous extract, the extract (1.25%, 2.5%, 3.75%, and 5%; w/v; water-basis) was previously dissolved in the water phase before adding the emulsifier mixture. The following steps were as previously described for the base emulsion preparation.

The A98C, G114C and Y143H mutations were introduced at the DNA level into plasmid pBIL-J30 using the QuikChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent Technologies, catalog No. 210515).
Wild-type J30 (J30 wt) and J30 CCH, each carrying a C-terminal polyhistidine tag, were produced in Escherichia coli NEB Express cells grown in TB medium with 50 µg ml⁻¹ kanamycin and 2 mM MgSO₄ at 200 rev min⁻¹. The bacterial suspensions were incubated at 37°C before induction with 0.5 mM IPTG at an OD_600 nm of 4 (J30 wt) or 1 (J30 CCH) and at 20°C thereafter. The protein expressions lasted 17 h (J30 wt) or 19 h (J30 CCH) until an OD_600 nm of 20 (J30 wt) or 20.5 (J30 CCH) was attained.
The cells were lysed in a buffer consisting of 0.15 M NaCl, 25 mM HEPES pH 7.4 with 1 mM PMSF using an Avestin EmulsiFlex-C3 homogenizer. The proteins were purified using an ÄKTAexplorer 100 Air by nickel-affinity (5 ml HisTrap HP or FF) and size-exclusion chromatography (Superdex 200 10/300 GL, all from GE Healthcare Life Sciences). All of the media and buffers used to produce and isolate J30 CCH also contained 10 µM ZnCl₂.

The cell pellet was resuspended in lysis buffer (30 mM TrisHCL pH 7.5; 300 mM NaCl; 10% glycerol, 2 mM MgCl₂, 2 µg/ml DNase I; 200 µg/ml lysozyme). The cells were lysed by passing three times through an EmulsiFlex-C3 (Avestin); the lysate was centrifuged (25000 × g; 30 min; 6 °C) supernatant was taken and spun again at 150000 × g; 1 h; 6 °C. The membrane pellets were collected and resuspended in high salt buffer (30 mM TrisHCl pH 7.5 800 mM NaCl; 10% glycerol) and ultracentrifuged (150000 rcf; 1:35 h; 6 °C). Membrane pellets were resuspended in 30 mM TrisHCl pH 7.5; 300 mM NaCl, 10% glycerol, flash frozen in liquid nitrogen and stored at −20 °C. Protein was solubilized by adding 1% DDM and followed by centrifugation at 50000 rcf for 30 min at 6 °C. Solubilized protein was first purified by a nickel-IMAC beads in a gravity column; washed with 10 mM and 30 mM imidazole in 30 mM TrisHCl pH 7.5; 300 mM NaCl, 10% glycerol; 0.03% DDM, and eluted with 250 mM imidazole. Elution fractions containing the protein were concentrated and loaded onto a gel filtration 10/300 S200 column in 20 mM HEPES pH 7.4, 150 mM NaCl and 0.03% DDM. Fractions containing the protein (5 ml) were concentrated in an Amicon® Ultra 4 ml centrifugal filter devices (50 kDa MWCO) to 0.5 ml, final concentration of 9 mg/ml and stored at 4 °C.

The cell pellet was resuspended in lysis buffer (30 mM TrisHCL pH 7.5; 200 mM NaCl; 5% glycerol, 2 mM MgCl₂, 2 µg/mL DNase I; 200 µg/ml lysozyme). The cells were lysed by passing three times through an EmulsiFlex-C3 (Avestin); the lysate was centrifuged (25000 × g; 30 min; 6 °C) supernatant was taken and spun again at 150000 × g; 1 h; 6 °C. The membrane pellets resuspended in buffer (30 mM TrisHCl pH 7.5; 200 mM NaCl, 5% glycerol), flash frozen in liquid nitrogen and stored at −20 °C. Protein was solubilized by adding 1% DDM and followed by centrifugation at 50000 rcf for 30 min at 6 °C. Solubilized protein was first purified by nickel-IMAC in a gravity column; washed with 40 mM imidazole in 30 mM TrisHCl pH 7.5; 200 mM NaCl; 5% glycerol; 0.03% DDM and eluted with 300 mM imidazole. Eluted fractions were combined with 1 mg of TEV protease/mg of protein to perform the His-tag cleavage during dialysis overnight at 4 °C. Protein solution was added to gravity nickel-NTA column to perform a reverse IMAC; the flow-through was collected, concentrated and loaded onto a gel filtration 10/300 S200 column in 20 mM HEPES pH 7.4, 150 mM NaCl and 0.03% DDM. Fractions containing the protein were concentrated in an Amicon® Ultra 4 ml centrifugal filter devices (100 kDa MWCO) to a final concentration of 2.5 mg/ml and stored at 4 °C.

An N-terminal His-tagged A_2AR was expressed in Pichia pastoris as described previously [18 ]. The receptor sequence terminated at Ala316 (Fig. S1) as this construct is degradation resistant [25 (link)] and has been used extensively in structural studies, having wild-type pharmacology [26 (link),27 (link)]. Prior to SMA-extraction, cells were disrupted following suspension in breaking buffer (50 mM sodium–phosphate buffer, 100 mM NaCl, 5% glycerol, EDTA-free protease inhibitor, pH 7.5, 4 °C) by 3–5 passes using an Avestin Emulsiflex C3 cell-disrupter. Unbroken cells and debris were removed by centrifugation (5000 ×g, 10 min, 4 °C). The A_2AR-expressing membrane fraction was then sedimented (100,000 ×g, 60 min, 4 °C) and re-suspended to 80 mg ml⁻¹ (wet weight) in extraction buffer (300 mM NaCl, 20 mM HEPES, pH 7.5). Membranes were stored at −80 °C until needed.