The protocol has been developed for the production of full-length huntingin variants at a low milligram scale. It allows for consistent high-quality protein essential to HD researchers. Demonstrating the procedure will be Michael Harris, a research scientist from the Curia Laboratory.
Begin by adding one gram of polyethyleneimine 25 K to one liter of endotoxin free-water, and stir well. Adjust the pH to 2.0 using 100-millimolar hydrochloric acid and stir until all the polyethyleneimine dissolves. Then adjust the pH to 7.0 using 100-millimolar sodium hydroxide solution and pass the solution through a 0.2-micrometer filter.
Make aliquots of the filtered solution and store them at minus 20 degrees Celsius. Propagate HEK293 cells in the growth medium supplemented with penicillin streptomycin in a humidified shaker incubator at 37 degrees Celsius, 90 RPM, 5%carbon dioxide for 18 to 24 hours. One day before the transfection, dilute the cells at a density of about 1.2 x 10 to the sixth power cells per milliliter in two liters of growth medium in a five-liter Erlenmeyer flask and incubate for 18 to 24 hours.
At the end of the incubation, measure the cell density and viability using an auto cell counter following the manufacturer's instructions. After calculating the amount of polyethyleneimine and plasmid required for transfection, dilute polyethyleneimine and plasmid individually in 100 milliliters of PBS and incubate at room temperature for five minutes. Then, mix the diluted plasmid and polyethyleneimine by gently swirling and incubate the mixture at room temperature for 30 minutes.
Add the mixture to the cell culture and mix by swirling gently. Now, allow the cells to grow for 24 hours. The next day, add sodium butyrate solution to a final concentration of two-millimolar anti-clumping and anti-foaming agent at 1:1, 000 volume ratio to the culture and incubate the cells in the humidified shaker incubator for 48 hours.
After measuring the cell viability and density, harvest the cells by centrifugation at 2, 000 G for 30 minutes and store the cell pellet at minus 80 degrees Celsius before purification. For anti-FLAG purification using FPLC, pack 12 to 25 milliliters of anti-FLAG resin onto an empty column at a flow rate of four milliliters per minute using buffer A and adjust the height of the plunger to remove the gap between the end of the plunger and the bed of resin. Next, suspend the thawed cell pellet in cold lysis buffer and pass the cell suspension once through a high-shear homogenizer at 1, 000 pounds per square inch pressure.
Using a centrifuge equipped with a compatible fixed-angle rotor, clarify the lysate at 20, 000 G for one hour. Program FPLC under the Method window, load the clarified lysate via the sample pump, and wash the column with the desired column volumes of buffer A, B, C, D, and elution buffer as described in the text manuscript. Once the run is finished, analyze 10 microliters of the peak fractions using SDS-PAGE.
Combine the peak fractions with the desired purity and save approximately 50 microliters of the combined eluates for further SDS-PAGE analysis. Start to equilibrate SEC column using the FPLC and load the anti-FLAG eluate via a 50-milliliter superloop. After running the SEC buffer, allow the SEC separation to occur overnight at four degrees Celsius.
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, and, if desired, pool the higher-ordered oligomeric and dimeric HTT fractions separately. Concentrate the pooled HTT protein using a 100-kilodalton centrifugal concentrator at four degrees Celsius.
After calculating the protein concentration, aliquot less than 100 microliters of the purified HTT protein in a cryo-safe microcentrifuge tube. Flash freeze the aliquots using liquid nitrogen and store them at minus 80 degrees Celsius. Perform all analytical SEC-MALS at four degrees Celsius on the HPLC system, coupled with a UV detector, a multi-angle light scattering detector, a differential refractive index detector, and use 0.185 as the refractive index increment for HTT.
Purge the pump and the detectors with filtered HPLC-grade water and connect the UHPLC column to the system. Equilibrate the column with filtered water and then SEC-MALS buffer until all the detector signals reach baseline. Inject two microliters of BSA solution at a flow rate of 0.3 milliliters per minute for 15 minutes per injection.
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. 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-micrometer spin filter.
Inject two to four microliters of the HTT sample and run for 15 minutes at four degrees Celsius at a flow rate of 0.3 milliliters per minute. Analyze the chromatographic and light scattering data using accompanying software and generate a Zimm plot to determine the weight-averaged molecular mass for each peak. In the present study, using a two-step column process, greater than 95%of purity was obtained for HTT and the major contaminant was chaperone Hsp70, which was eliminated by extensive washing with magnesium chloride and ATP during the anti-FLAG affinity purification step Overexpression of FL HTT can result in fragmentation of the protein but FL Q23-HTT produced by the method resolved as a single band with the correct molecular weight of 350 kilodaltons indicates no fragmentation.
Western blot analysis after the reaction of antibodies with FL Q23-HTT demonstrated that the protein was isolated without significant detectable truncations, as any additional fragment-related bands were not observed. FL HTT poly-Q length variance, Q23, Q48, and Q73 reacted as expected in Western blot, showing a progressively stronger signal for poly-Q-directed monoclonal antibody MW1, correlating with increasing Q length. Among the HPLC columns tested, the SEC column showed sufficient resolution between the HTT monomer and dimer, such that molar masses could be distinguished.
Purified FL HTT from SEC showed multiple bands corresponding to the oligomerization states, which could be because of the presence of several hydrophobic patches which contribute to the formation of higher-ordered oligomers during migration within the gel. Purified HTT showed three major bands on blue native PAGE with an estimated molecular weight of 643, 927, and 1, 070 kilodaltons that likely represent the monomeric, dimeric, and trimeric species of HTT, respectively. Proper handling of purified huntingtin is essential to avoid generating high-ordered oligomers in soluble aggregates.
It is imperative the end user works quickly to dispense the sample into pre-chilled tubes before flash freezing. Long-term stability testing can be performed on samples stored at extended periods at minus 80 degrees Celsius. Repeat HPLC SEC-MALS analyses will confirm if there has been any change to the sample's monomeric content.
