Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

1. Transfection of Adherent HEK293S GnTI^– Cells for Thermostability Screen

1. Prepare Poly-D-Lysine (PDL)-coated 96-well plates. Perform all work in a sterile laminar flow hood.
   1. Filter a 25 µg/mL solution of (PDL), molecular weight 70,000-150,000 Da, using a 0.2 µm sterile filter. Add 50 µL of PDL to each well of a 96-well Tissue Culture (TC) plate, ensuring the bottom is evenly coated, and incubate at RT for 30 min.
   2. Aspirate PDL solution and wash with 200 µL of sterile water.
   3. Allow plate to air dry for 2 h prior to storage at 4 °C. PDL coated plates can be stored at 4 °C for several weeks.
2. Trypsinization of adherent HEK293S cells.
   1. Grow HEK293S to 80% confluency in a 10 cm dish maintained at 37 °C with 8% CO₂. A single 10 cm dish should have approximately 10 million HEK293S cells. Do not use cells after 30 passages!
   2. Aspirate media and wash with 5 mL of Phosphate-Buffered Saline (PBS). Add 1 mL of Trypsin-EDTA solution (0.25% trypsin, 0.02% EDTA) to cells and incubate for 2 min at 37 °C.
   3. Add 10 mL of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and resuspend cells by repeatedly pipetting up and down using a serological pipette.
   4. Pipette 10 µL of cells and mix with 10 µL of Trypan Blue solution (0.4%). Determine cell density using a hemocytometer and a light microscope. Do not count cells that are stained blue as they are dead. Ensure that cell viability is above 90%.
3. Thoroughly resuspend trypsinized HEK293S GnTI^– cells in DMEM supplemented with 10% FBS to a density of 0.5 x 10⁶ cells/mL in a disposable pipette reservoir. Approximately 5 million HEK293S cells will be needed for each plate. A total of 24 constructs can be transfected on each plate using this protocol.
4. Using a multichannel pipette, add 100 µL of cells to each well of a PDL coated plate. Resuspend cells in the pipette reservoir after filling each plate to ensure an even distribution of cells. Incubate cells in a 37 °C incubator with 8% CO₂. After 24 h, cells should reach approximately 80% confluency.
5. 1 h prior to transfection replace media. Prepare DNA-transfection reagent complexes for transfection.
   1. For each construct in the SERT_TC background to be screened, mix 450 ng DNA with 45 µL of serum-free DMEM and mix. Add 1.6 µL of the transfection reagent to 45 µL of serum-free DMEM and mix.
   2. Immediately add diluted transfection reagent solution to DNA solution and mix. Do not mix solutions in the reverse order. Wait 10 – 15 min and add 20 µL of transfection reagent/DNA mixture to 4 wells.
6. Once all wells have been transfected, mix plate by gently rocking back and forth and return to the 37 °C incubator.
7. After 16 – 24 h replace media with DMEM containing serum and 10 mM sodium butyrate.
8. Approximately 48 h post transfection remove media and either immediately proceed with thermostability screen or freeze cells at -80 °C to be used later. Cells can be stored at -80 °C for several weeks.

2. Scintillation Proximity-based Thermostability Screen with S-citalopram

1. Incubate cells with 200 nM S-citalopram in 25 µL of TBS (Tris-Buffered Saline — 20 mM Tris, pH 8, 100 mM NaCl) for 5 min at RT.
2. Solubilize cells by adding 25 µL of 8 mM n-dodecyl-β-D-maltopyranoside (C12M), 1 mM cholesteryl hemmisuccinate (CHS), protease inhibitor cocktail (2 mM phenylmethanesulfonyl fluoride (PMSF), 0.1 mg/mL aprotinin, 4 g/mL pepstatin A, and 4 µg/mL leupeptin) in TBS, and incubating for 1 h at RT.
3. Add 50 µL of TBS with 20 nM [³H]citalopram (81.7 Ci/mmol), 0.1% bovine serum albumin, and 2 mg/mL His-tag affinity scintillation proximity assay (SPA) beads to three wells for each construct. For the last well add the same solution listed but supplement with 100 µM sertraline to determine nonspecific binding. Ensure that His-tag affinity SPA beads are thoroughly mixed when adding to the 96-well plate.
4. Measure [³H]citalopram binding using a 96-well scintillation counter at RT with a 1 min count time per well. Continue counting plates until total counts plateau (approximately after 36 h).
5. Heat plates for 15 min in a heating block with a heated lid at 33 °C. Measure [³H]citalopram binding again after heating as described in 2.4.
6. Repeat step 2.4 – 2.5 and change the heating step to 36 °C, 39 °C, 45 °C, 48 °C, and 51 °C. Adjust heating temperatures depending on the apparent melting temperature (Tm) of the target protein and the thermostability of the most stable construct. Continue to heat plates until all constructs have low specific counts.
7. Analyze data to determine Tm values.
   1. Determine the average total counts per min (CPM) of each construct for wells that do not contain sertraline by averaging the 3 replicate wells. Determine the nonspecific CPM by averaging the CPM of each well containing sertraline in a single plate.
   2. Calculate specific CPM by subtracting the nonspecific CPM from the total CPM.
   3. Calculate the Tm by a non-linear fit to a Boltzmann sigmoidal function. Constructs with low specific CPM at RT (<10% of WT) generally do not have accurate Tm values. Mutations from the most thermostable constructs can be combined together and screened for additive increases in thermostability.

3. Expression of the Human Serotonin Transporter in HEK293S GnTI^– Cells

1. Transform chemically competent DH10Bac cells with 1 ng of plasmid.
   1. Add plasmid to 50 µL of DH10Bac cells and incubate on ice for 30 min.
   2. Heat shock DH10Bac cells at 42 °C for 30 sec. Add 200 µL of SOC medium and incubate at 37 °C for 4 h with shaking.
   3. Plate all of the bacteria onto a Luria Broth (LB) plate containing 50 µg/mL kanamycin, 7 µg/mL gentamicin, 10 µg/mL tetracycline, 100 µg/mL 5-bromo-3-indolyl β-D-galactopyranoside, and 40 µg/mL isopropyl β-D-1-thiogalactopyranoside (IPTG).
2. Isolate lacZ^– colonies (white colonies) grown at 37 °C for 2 d on LB agar plates.
3. Grow several colonies O/N at 37 °C in 5 mL of LB containing antibiotics and isolate bacmid DNA.
   1. Spin down bacteria at 1,000 x g in a centrifuge for 5 min. Discard supernatant. Resuspend bacteria with 200 µL of miniprep resuspension buffer.
   2. Lyse bacteria by adding 200 µL of miniprep lysis buffer and inverting tube gently 10 times. Add 200 µL of neutralization buffer.
   3. Remove insoluble fraction by spinning at 14,000 x g for 10 min in a centrifuge. Add 1 mL of isopropanol to supernatant and chill at -20 °C for 20 min to precipitate DNA.
   4. Spin at 14,000 x g for 15 min in a centrifuge and discard supernatant.
   5. Wash DNA pellet with 70% EtOH and spin again at 14,000 x g for 15 min. Discard supernatant. Air dry DNA until all EtOH has evaporated and resuspend in 50 µL of water.
      NOTE: Bacmid DNA should be transfected into Sf9 cells immediately for best results but may also be stored at -20 °C for several weeks.
4. Transfect bacmid DNA into 1×10⁶ cells of adherent Sf9 grown in a humidified chamber at 27 °C in a 6-well dish. Perform all cell culture manipulations in a sterile laminar flow hood.
   1. Remove media from cells and add 2 mL of fresh Sf9 media. Add 5 µg of bacmid DNA to 100 µL of Sf9 media (Solution A).
   2. Add 8 µL of a cationic-lipid Sf9 transfection reagent to 100 µL of Sf9 media (Solution B). Incubate tube containing Solution B for 5 min.
   3. Mix tube containing Solution A with Solution B and incubate at RT for 30 min and add all of the solution to the Sf9 cells.
   4. After 96 h, harvest supernatant (P1 virus) by passing through a 0.2 µm filter. The P1 virus may be stored for several months at 4 °C in the dark and reused to make P2 virus as needed.
5. Add 100 µL of P1 virus to 1 L of Sf9 cells at a density of 1 x 10⁶ cells/mL in Sf9 media. Infect cells for 96 h, growing at 27 °C on a shaker at 100 rpm.
6. Spin down cells in a centrifuge at 4,000 x g for 15 min and filter supernatant containing virus particles through a 0.2 µm filter. Discard cell pellet. Determine viral density using a viral plaque assay or a virus counter. The virus density should be >1 x 10⁸ virus particles per milliliter. P2 virus can be stored at 4 °C in the dark and used for several months.
7. Infect 10 L of HEK293S GnTI^– cells⁷ growing in suspension at 37 °C with 8% CO₂ and 85% humidity on a shaker at 130 rpm in 293 expression media supplemented with 2% FBS at a multiplicity of infection (MOI) of 2 and a density of 3 x 10⁶ cells/mL, typically 30 – 50 mL of P2 virus per 800 mL of cells in a 2 L baffled flask.
   NOTE: It is not recommended to use more than 80 mL of P2 virus since the HEK293S GnTI^– cells will grow slowly and may become unviable due to a change in pH. Sf9 media is more acidic than the 293 expression media.
8. 12 – 16 h post-infection, add sodium butyrate to a concentration of 10 mM from a 1 M stock. 48 – 60 h post-infection, harvest cells by centrifugation at 4,000 x g for 15 min. Remove the supernatant.
9. Resuspend cells in 150 mL of TBS, 2 µM S-citalopram or other SERT inhibitors and store at -80 °C until ready for purification.

4. Affinity Purification of the Serotonin Transporter for Immunization and Crystallization

1. Thaw cells from 10 L of culture in warm water (approximately 30 °C) and resuspend by rapidly passing through a 10 mL pipette until homogeneous.
2. Prepare detergent solution for solubilization (150 mL): 80 mM Tris, pH 8, 150 mM NaCl containing 40 mM C12M, 5 mM CHS, and protease inhibitor cocktail.
3. Add all of the cells to a beaker with a stir bar and add all of the detergent solution to the cells while stirring. Solubilize at 4 °C for 1 h with stirring.
4. Spin lysate at 8,000 x g for 15 min at 4 °C. Discard pellet and decant supernatant into clean ultracentrifuge tubes. Spin at 100,000 x g for 1 h in an ultracentrifuge. Discard pellet and filter supernatant through a 0.2 µm filter.
5. Pass lysate over 10 mL of Strep affinity resin packed into a column using a peristaltic pump equilibrated in wash buffer: 1 mM C12M, 0.2 mM CHS, 5% glycerol, 25 µM lipid (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol at a molar ratio of 1:1:1), and 1 µM S-citalopram or SSRI in TBS.
6. Connect Strep affinity column to a Fast Protein Liquid Chromatography (FPLC) system and wash column at 2 mL/min with 66 mL (6.6 column volumes) of wash buffer. Elute purified protein in same buffer supplemented with 5 mM desthiobiotin at 0.5 mL/min using 33 mL (3.3 column volumes) of buffer. Collect 1 mL fractions; the peak fractions will be ~10 mL. Purified protein may be stored at 4 °C for 2 – 3 d if desired.
   NOTE: Total yield will be 3 – 4 mg of SERT_CC and 1 mg of SERT_IC. An absorbance of 2 AU at 280 nm is equal to 1 mg/mL SERT.

5. Reconstitution of Transporter into Liposomes for Immunization

1. Prepare liposomes containing asolectin:cholesterol:lipid A:brain polar lipid (molar ratio 60:17:3:20). Dissolve lipids in 1 mL of chloroform and add 40 mg of total lipid to a 100 mL glass round bottom flask at the indicated molar ratio.
   1. Evaporate chloroform for at least 1 h under vacuum with the round bottom flask rotating in warm water, approximately 30 °C.
   2. Rehydrate lipids by adding 10 mL of TBS. Incubate in buffer for 10 min.
   3. Freeze the lipid by placing the flask in liquid N₂.
   4. Thaw. Immerse spherical bottom of the flask in a beaker filled with warm water, approximately 30 °C. Sonicate in a bath sonicator for 5 min.
   5. Vortex and repeat steps 5.1.3 – 5.1.4 10 times or until the lipid is completely resuspended from the bottom and forms a cloudy suspension.
   6. Extrude the entire lipid mixture twice through 200 nm filters. The lipid will appear as a milky suspension prior to extrusion; afterward it will be translucent. Do not add the entire lipid mixture to the extruder at once as it can become clogged, resulting in loss of sample.
   7. Spin lipids at 100,000 x g for 20 min and resuspend in 0.5 – 1 mL of TBS at a concentration of 40 mg/mL.
2. Concentrate purified SERT_IC to 250 – 500 µL using a 100 kDa MWCO centrifuge protein concentrator to 2 – 4 mg/mL and saturate liposomes with 5 mM C12M. Add purified SERT to the detergent:lipid mixture in 1 mL final volume.
3. Remove C12M by 3 successive additions of 80 mg/mL hydrophobic absorption resin. For the first 2 additions, incubate with resin with rotation for 2 h at 4 °C. Remove resin by passing through glass wool. Perform the final incubation with resin overnight.
4. Concentrate proteoliposomes by centrifugation at 100,000 x g for 20 min. Discard supernatant and resuspend pellet in 250 – 500 µL of TBS.
5. Add 10 µM final concentration of S-citalopram to the reconstituted protein following the final removal of resin.
6. Solubilize 2.5 µL of the proteoliposomes in SDS-PAGE loading buffer (62.5 mM Tris, pH 6.8, 10% glycerol, 2% SDS, 0.01% bromophenol blue, 100 mM DTT) and run on a SDS-PAGE gel at 200 V for 1 h to ensure that SERT has been successfully reconstituted. Proteoliposomes can be stored at -80 °C in aliquots for long term storage and thawed on ice prior to each immunization.

6. Screening for Antibodies Recognizing 3D Epitopes

1. Screen hybridoma cell lines by western blot. Antibodies which recognize SERT by western blot likely bind linear epitopes and will likely not be useful for structural studies.
   1. Mix 1 µg of purified SERT_IC or SERT_CC and run on a 4 – 15% SDS-PAGE gel at 200 V for 1 h.
   2. Transfer to a nitrocellulose membrane at 200 mA for 30 min using Towbin buffer (25 mM Tris, 192 mM glycine, 20% (v/v) methanol, 0.1% SDS). The membrane can be dried and stored indefinitely at room temperature.
   3. Rewet membrane with 10% methanol, and wash with PBS (10 mM phosphate, pH 7.4, 137 mM NaCl, 2.7 mM KCl).
   4. Block with 5% powdered milk in PBS for 30 min at RT.
   5. Wash with PBS and incubate with 1 µg/mL of antibody in PBS with 0.1% milk.
   6. Wash extensively with PBS containing 0.1% Tween-20.
   7. Incubate with goat anti-mouse antibody conjugated to an IR dye diluted 1:10,000 in PBS with 0.1% Tween 20 and 0.1% milk.
   8. Wash extensively and scan using an imaging system.
2. Mix hybridoma supernatant containing western negative antibodies with 100 nM SERT_CC protein using a molar ratio of 1:2 (SERT:mAb) in 200 µL TBS with 1 mM C12M, 0.2 mM CHS, and 1 µM S-citalopram. Centrifuge at 100,000 x g for 20 min. Analyze supernatant containing SERT-mAb complexes and analyze by Fluorescence-detection Size Exclusion Chromatography (FSEC)⁸. Discard the pellet.
   1. Run 100 µL of supernatant on a size exclusion column at 0.5 mL/min using a High Performance Liquid Chromatography (HPLC) system equipped with a fluorescence detector (Excitation: 480 nm, Emission: 510 nm) using a running buffer containing TBS, 0.4 mM lauryl maltose neopentyl glycol, and 1 µM S-citalopram. Antibodies which form complexes will cause the GFP-fusion protein to elute earlier than the free transporter from the size-exclusion column.
   2. Dilute complexes to 10, 1, 0.1 nM in 200 µL TBS with 1 mM C12M, 0.2 mM CHS, and 1 µM S-citalopram and rerun FSEC. Antibodies which can still shift the transporter peak at low nanomolar concentrations are high affinity binders and good candidates for structural studies.
3. Retest binding by FSEC in the presence of 1 mM serotonin. Antibodies which specifically recognize the SSRI bound conformation will not bind in the presence of the substrate. Antibodies which recognize a 3D epitope which does not change as a result of conformation will bind regardless of the ligand present.
4. Mix pairwise different combinations of antibodies and test binding by FSEC. Antibodies which recognize distinct epitopes will cause SERT to elute earlier when combined together compared to binding of a single antibody.
5. For initial structural studies, select the highest affinity antibodies which recognize 3D epitopes.

7. Expression of Antibody Fragment in Sf9 Cells

1. Clone variable and constant domains of Fab by PCR into the insect expression system for expression in Sf9 cells using standard protocols.
2. Transfect 1 x 10⁶ Sf9 cells with 5 µg of Bacmid DNA encoding the light and heavy chains of the 8-His tagged 8B6 Fab with a GP67 secretion sequence (as per section 3.4).
3. Harvest P1 virus 96 h posttransfection and add 500 µL of P1 virus to 1 L of Sf9 cells at a density of 1 x 10⁶ cells/mL in a 2 L flask to make P2 virus. Infect cells for 96 h at 27 °C.
4. Harvest P2 virus (as per section 3.6).
5. Infect 6 L of Sf9 cells at a density of 2 – 3 x 10⁶ cells/mL with an MOI of 2 with P2 virus, typically 40 – 50 mL per flask with 1 L of Sf9 cells in each 2 L flask.
6. Harvest cells approximately 96 h postinfection. Add 50 mL of phosphate buffer, pH 8 at a final concentration of 50 mM to the cells and spin at 4,000 x g for 20 min. Discard the cell pellet and filter supernatant through a 0.2 µm filter tangential flow cell. Collect supernatant and store at 4 °C for 2 – 3 days if desired.

8. Purification of Antibody Fragments from Sf9 Supernatant

1. Concentrate Sf9 supernatant using a 30 kDa Molecular Weight Cut Off (MWCO) tangential flow cell to approximately 400 – 800 mL.
2. Add imidazole, pH 8 at a final concentration of 10 mM and 10 mL of His-tag affinity resin. Stir in a beaker for 1 h at 4 °C.
3. Collect His-tag affinity resin by centrifugation at 2,000 x g for 5 min. Discard supernatant.
4. Pack His-tag affinity resin into a column and connect to an FPLC.
5. Wash His-tag affinity resin at 2 mL/min with 66 mL (6.6 column volumes) of 50 mM phosphate pH 8, 150 mM NaCl, 25 mM imidazole.
6. Elute 8B6 Fab in 33 mL of 50 mM phosphate pH 8, 150 mM NaCl, 250 mM imidazole. Collect 1 mL fractions.
7. Dilute affinity purified Fab with a 10-fold volume of ice-cold 20 mM acetate, pH 5.
8. Bind Fab to a 1 mL cation exchange column using a peristaltic pump at 1 mL/min.
9. Separate Fab using a 30 mL linear gradient of NaCl (0 – 500 mM) in 20 mM acetate pH 5.5 using an FPLC. Fab will elute as a single peak at ~300 mM NaCl.
10. Analyze on a 12.5% SDS-PAGE gel using sample buffer containing 100 mM DTT. The heavy and light chains of the 8B6 Fab will run at 27 and 25 kDa, respectively, on a reducing SDS-PAGE gel.
11. Pool fractions containing Fab and concentrate to at least 10 – 15 mg/mL using a 30 kDa MWCO protein concentrator in a swinging bucket centrifuge at 3,000 x g.
12. Adjust to pH 8 by adding 1 M Tris pH 8 to a final concentration of 50 mM and store purified Fab for long term at 4 °C. Total yield will be 25 – 30 mg. An absorbance of 1.4 AU at 280 nm is equal to 1 mg/mL of 8B6 Fab.

9. Formation of Transporter-antibody Complexes and Separation by Size Exclusion Chromatography

1. For crystallization, purify the SERT_CC by Strep affinity chromatography in C12M as described earlier (section 4).
2. Digest with thrombin to remove tags (1:100 w/w) and EndoH (1:10 w/w) O/N at RT. Concentrate to 250 – 300 µL using a 100 kDa MWCO centrifuge protein concentrator to a protein concentration of 10 mg/mL.
3. Mix concentrated SERT with Fab at a molar ratio of 1:1.2 in a volume less than 500 µL. Centrifuge at 100,000 x g at 4 °C for 20 min. Collect supernatant containing SERT-Fab complex. Discard pellet.
4. Separate by Size Exclusion Chromatography (SEC) using an FPLC on a size exclusion column equilibrated in TBS supplemented with 40 mM n-octyl-β-D-maltoside (C8M), 0.5 mM CHS, 5% glycerol, 25 µM lipid (same as in step 4.5), and 1 µM S-citalopram at 0.5 mL/min.
5. Collect 0.5 mL fractions and analyze on 4 – 15% SDS-PAGE gels as well as by tryptophan fluorescence by SEC. GFP tagged SERT will run at approximately 80 kDa on a SDS-PAGE gel. After removal of the termini and N-linked sugars it will run as a 45 kDa protein.
6. Determine which fractions to combine by pooling only the fractions which are monodisperse as judged by analysis of the fractions by tryptophan fluorescence SEC (Excitation: 280, Emission: 335 nm).
7. Store purified SERT-8B6 complex at 4 °C for up to 1 week.

10. Crystallization of Transporter-antibody Complexes by Hanging Drop

1. Prior to crystallization, concentrate peak fractions from the SEC separation of the SERT-8B6 complex to 2 mg/mL using a 100 kDa MWCO centrifuge protein concentrator. An absorbance of 2 AU at 280 nm is equal to 1 mg/mL.
2. Add additional Fab at a ratio of 1:0.05 complex:free Fab. Add 10 µM free S-citalopram.
3. Centrifuge at 100,000 x g at 4 °C for 20 min. Collect supernatant containing the SERT-Fab complex. Discard pellet.
4. Set up a hanging drop 24-well screen at 4 °C according to Table 1. Grow diffraction quality crystals over reservoir solutions containing 100 mM Tris-NaOH, pH 8.5, 25 – 125 mM KCl, 32.5 – 34% PEG 400, and 0.5% 6-aminohexanoic acid.
   NOTE: Use Tris adjusted with NaOH to pH 8.5 as a buffer. Do not use Tris base adjusted with HCl. The screen may be prepared in 2 mL tubes and used until finished. Take care to accurately pipette PEG 400 using a positive displacement pipette as it is extremely viscous!
   1. Pipette 500 µL of each reservoir solution in a low profile 24-well plate with sealant applied to each well. Pipette 1.5, 1.75, and 2 µL of the SERT-8B6 complex onto an 18 mm siliconized glass cover slip. Pipette 1 µL of reservoir solution on top of the protein sample.
      NOTE: The plate was purchased with sealant already applied to the rim of the wells.
   2. Apply cover slide to the 24-well with the drops facing the reservoir solution and seal immediately by pressing cover slip onto sealant pre-applied to each well.
   3. Continue until all 24-wells have been set up.
   4. Leave the finished plate in a well-insulated room at 4 °C. Do not disturb plates for at least 3 days
      NOTE: Single crystals will appear within approximately 3 days and grow to 100-175 µm after 14 days.
5. Harvest crystals in cryoloops and directly flash-cool in liquid N₂ prior to X-ray diffraction data collection.
6. If necessary, find additional crystallization conditions by broad screening with hanging drops. Use three drops with protein:precipitant ratios of 2:1, 1.5:1, 1:1. If a robot is available, then set up 100-150 nL drops over 70 µL of reservoir solution in a 96-well plate. Larger 3D crystals can be grown in 24-wells.
7. Score based on the appearance of the drop: 0, clear; 1, dust; 2, granular precipitate; 3, phase separation; 4, microcrystalline; 5, needles; 6, plates; 7, 3D crystals.
8. Set up crystallization by hanging drop methods in 24-wells as described above around newly identified conditions.