This method allows us to produce tag-free exon one bearing the native sequence of the protein and provides new opportunities for studying the structure function of this protein, thus enabling us to understand its function in health and disease. The main advantages here is the simplicity, efficiency, and robustness of the method. By eliminating the heterogeneity of the sequence, this method will enable and facilitate comparison and reproducibility across different laboratories.
Though this method was developed for the production of Huntingtin exon one, it can also be applied to other aggregation-prone systems like amyloid-beta peptide and alpha-synuclein. First, filter a previously isolated supernatant containing His-SUMO Huntingtin exon one fusion protein using a 0.45-micrometer syringe filter. Remove a 20-microliter sample of the filtered supernatant for SDS-PAGE analysis.
Fill the clarified lysate into a Superloop. Isolate the His-SUMO Huntingtin exon one fusion protein from the clarified bacterial lysate by immobilized metal affinity chromatography on a fast performance liquid chromatography system at four degrees Celsius. Pass 10 column volumes or 200 milliliters of buffer A at 10 milliliters per minute to wash out the unbound proteins.
Elute the fusion protein with 2.5 column volumes or 50 milliliters of 100%buffer B at two milliliters per minute. Take 20 microliters of each fraction for SDS-PAGE analysis. Add 20 microliters of loading dye to each sample.
Load two microliters of the crude protein, the unbound fraction, the wash fraction, and each fraction of the elution peak on a 15%gel. Run the gel for 90 minutes at 180 volts. When the run is finished, stain the gel with Coomassie dye according to the manufacturer's instructions.
Pool the fractions containing the fusion protein according to the peak of the chromatogram. Then add 100-millimolar DTT and 100-millimolar L-cysteine to the pooled fractions, and dissolve by gently inverting the tube. To prepare solvent A for HPLC purification, add two milliliters of trifluoroacetic acid to a two-liter bottle of water.
To prepare solvent B, add 2.5 milliliters of trifluoroacetic acid to a 2.5-liter bottle of acetonitrile. Prepare the HPLC system as suggested by the manufacturer. Perform a blank run to ensure a clean column.
Following this, take 100 microliters of the fusion protein before the addition of ULP1 to monitor the cleavage reaction by UPLC. Transfer 20 milliliters of the fusion protein to a new 50-milliliter tube, and add 0.4 milliliters of ULP1 stock solution. Incubate the sample on ice, and keep the remaining fusion protein on ice.
Do not store the protein during intermediate steps, and proceed with the purification until completion. Every 10 minutes, remove 100 microliters of the cleavage reaction to monitor the progress of UPLC. After centrifuging the samples, analyze two microliters of each supernatant by UPLC.
Compare the chromatograms obtained for the sample before addition of ULP1 and the samples taken after. Once the SUMO cleavage is complete, filter the sample with a 22-micrometer syringe filter. Next, purify the filtered sample by reversed phase HPLC.
Analyze the HPLC fractions by UPLC and ESI-MS. Pool fractions of similar purity in 50-milliliter plastic tubes. Freeze the samples in liquid nitrogen and lyophilize.
Then transfer the lyophilized protein into two-milliliter plastic tubes, and store at minus 20 degrees Celsius. Dissolve 100 micrograms of lyophilized Huntingtin exon one in eight microliters of neat trifluoroacetic acid in a 1.5-milliliter tube. After incubating for 20 minutes at room temperature, carefully evaporate the trifluoroacetic acid under a fume hood with a stream of nitrogen or argon.
Once the protein is dry, dissolve it in 100 microliters of water. Then analyze two microliters of sample by UPLC. Now mix 20 microliters of protein solution with 20 microliters of 2X loading dye.
Analyze one-to 10-microgram samples of protein by SDS-PAGE. Run the gel for 90 minutes at 180 volts. When the run is finished, stain the gel with Coomassie dye according to the manufacturer's instructions.
Prepare 10 milliliters of DPBS in a 50-milliliter tube. Filter the DPBS solution through a 2-micrometer filter before each use. Following this, dissolve 150 microliters of lyophilized Huntingtin exon one in 12 microliters of neat trifluoroacetic acid in a 1.5-millimeter tube.
After incubating for 20 minutes at room temperature, carefully evaporate the trifluoroacetic acid in a fume hood with a stream of nitrogen or argon. Dissolve the disaggregated protein in one milliliter of pre-cooled DPBS, and adjust the pH between 7.2 and 7.4 with one molar sodium hydroxide. Then filter the protein solution through a 100-kilodalton centrifugal filter into 1.5-milliliter plastic tubes.
Determine the concentration of Huntingtin exon one using a UPLC calibration curve based on amino acid analysis detection. Then calculate the amount of DPBS that needs to be added to obtain a concentration of three-micromolar Huntingtin exon one. Dilute the protein to three micromolar by adding the calculated amount of DPBS to the test tube.
Keep the tube on ice until initiation of the aggregation protocol. The validation by electro-microscopy and other technique has to be performed in order to validate that the disaggregation protocol was successful in removing any preformed aggregates. The complete amino acid sequence of the expressed fusion protein is shown here.
A schematic of the plasmid and expressed fusion protein is displayed here. His-SUMO Huntingtin exon one expresses at a medium level, and most of the fusion protein is present in the soluble fraction after lysis, both for the 23 glutamines and 43 glutamine variant. Both the wild-type and mutant fusion protein can be enriched to approximately 80%purity by immobilized metal affinity chromatography.
Cleavage of the His-SUMO tag and purification of Huntingtin exon one are shown here. The original fusion protein peak is consumed, and two new and well-separated peaks corresponding to the His-SUMO tag and Huntingtin exon one appear. Both Huntingtin exon one 23 glutamines and 43 glutamines can be separated from the His-SUMO tag by reversed phase HPLC and were obtained in high purity as shown by UPLC, MS, and SDS-PAGE analysis.
The aggregation kinetics of m-Huntingtin exon one fibril formation as determined by a sedimentation assay shows complete depletion of soluble protein after 48 hours of incubation. CD spectroscopy of the protein secondary structure shows a shift of Huntingtin exon one 43 glutamines from unstructured to the beta-sheet-rich conformation. This structural change is accompanied by the formation of long fibrillar aggregates as observed by TEM.
Once mastered, this method for the expression and purification of Huntingtin exon one can be carried out in three days if it's performed properly. While attempting this procedure, it's very important to remember that Huntingtin exon one proteins, especially protein with expanded polyglutamine stretch, have a very high propensity to aggregate. Thanks to this method, we're able to produce sufficient Huntingtin exon one proteins and perform previously inaccessible studies to investigate the structure, aggregation, and toxicity of the protein and to develop new assays to detect and quantify disease-associated form of Huntingtin exon one.
The stability and high solubility of the SUMO fusion protein provides greater flexibility in manipulating the protein and allows introduction of modification in the context of the fusion proteins that would otherwise not be possible after cleavage. We hope that the simplicity and efficiency of this method will eliminate the need of using nonnative sequences and model systems and encourage researcher from different discipline to join our quest to understand the biology of this important protein and develop effective therapeutics for the treatment of Huntington disease.
