High-titer recombinant baculovirus (109 viral particles per milliliter) was obtained using the Bac-to-Bac Baculovirus Expression System (Invitrogen). Sf9 cells at a cell density of (2–3) × 106 cells ml−1 were infected with the virus at a multiplicity of infection of 10 with the addition of 8 μM zafirlukast (Cayman Chemical). Cells were harvested by centrifugation at 48 hours after infection and stored at −80°C until use.

Insect cell membranes were disrupted by thawing frozen cell pellets in a hypotonic buffer containing 10 mM Hepes (pH 7.5), 10 mM MgCl2, 20 mM KCl, and protease inhibitor cocktail [PIC; 500 μM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (Gold Biotechnology), 1 μM E-64 (Cayman Chemical), 1 μM leupeptin (Cayman Chemical), 150 nM aprotinin (AG Scientific)] with the ratio of 50 μl per 100 ml of lysis buffer. Extensive washing of raw membranes was performed by repeated centrifugation for 30 min at 220,000g at 4°C and resuspension in the same buffer and then in a high-salt buffer containing 10 mM Hepes (pH 7.5), 10 mM MgCl2, 20 mM KCl, 1 M NaCl, and PIC (50 μl per 200 ml of lysis buffer) (two and three times, respectively). Purified membranes were then resuspended in the presence of 25 μM zafirlukast or pranlukast, iodoacetamide (2 mg ml−1), and PIC (50 μl per 50 ml of resuspension buffer) and incubated at 4°C for 30 min before solubilization. Receptor was extracted from the membrane using 1% (w/v) n-dodecyl-β-d-maltopyranoside (DDM; Avanti Polar Lipids) and 0.2% (w/v) cholesteryl hemisuccinate (CHS; Sigma-Aldrich) with continuous stirring at 4°C for 3.5 hours. The supernatant was isolated by centrifugation at 460,000g for 45 min at 4°C and incubated with TALON IMAC (immobilized metal affinity chromatography) resin (Clontech) overnight at 4°C in the presence of 10 mM imidazole.

The resin was then washed at 4°C with six column volumes (CVs) of 100 mM Hepes (pH 7.5), 250 mM NaCl, 10% (v/v) glycerol, 0.1% (w/v) DDM, 0.02% (w/v) CHS, 15 mM imidazole, PIC (50 μl per 100 ml of buffer), 10 mM MgCl2, and 8 mM adenosine 5′-triphosphate, and with six CVs of 50 mM Hepes (pH 7.5), 250 mM NaCl, 10% (v/v) glycerol, 0.05% (w/v) DDM, 0.01% (w/v) CHS, 30 mM imidazole, and PIC (50 μl per 100 ml of buffer). Then, the buffer was replaced with 50 mM Hepes (pH 7.5), 250 mM NaCl, 10% (v/v) glycerol, 0.05% (w/v) DDM, 0.01% (w/v) CHS, and 10 mM imidazole, and CysLT1R was treated with PNGase F (Sigma-Aldrich) for 4.5 hours to deglycosylate the receptor. The protein was then eluted with 5 CVs of 50 mM Hepes (pH 7.5), 250 mM NaCl, 10% (v/v) glycerol, 0.015% (w/v) DDM, 0.003% (w/v) CHS, and 300 mM imidazole. A PD-10 desalting column (GE Healthcare) was used to remove imidazole. The protein was then treated overnight at 4°C with His-tagged TEV protease (home-made) to remove the N-terminal Flag and His tags. The TEV protease and the cleaved 10× His tag were removed by incubating the sample for 1.5 hours with TALON IMAC resin. The receptor was then concentrated to 50 to 60 mg ml−1 with a 100-kDa molecular weight cutoff concentrator (Millipore). In the case of crystallization with zafirlukast, 50 μM zafirlukast (Sigma-Aldrich) was added to the elution buffer, 200 μM after the desalt procedure, and 10 μM into the washing buffers. In the case of crystallization with pranlukast, 50 μM pranlukast (Sigma-Aldrich) was added to the elution buffer and after the desalt procedure and 10 μM into the washing buffers. Protein purity and monodispersity were tested by SDS–polyacrylamide gel electrophoresis and analytical size exclusion chromatography (aSEC). Typically, protein purity exceeded 95%, and aSEC profiles showed a single peak with less than 10% aggregation level (fig. S1A). Protein stability was assessed by a microscale thermoshift assay using a 10 μM CPM [7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin, Invitrogen; λex = 384 nm; λem = 470 nm] dye on a Rotorgene-Q (QIAGEN) instrument (37). Typical melting temperatures were higher than 70°C (fig. S1B).

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.