Purification of Alpha-Synuclein
By Dr. Zubair Khalid, DVM, MS, PhD ·
Quick Answer
- Purify alpha-synuclein using anion exchange chromatography as the primary capture step, exploiting the protein's acidic isoelectric point near pH 4.7.
- Follow anion exchange with size exclusion chromatography to separate monomeric alpha-synuclein from preformed oligomers and aggregates.
- Maintain cold temperatures and avoid freeze-thaw cycles because alpha-synuclein is intrinsically disordered and prone to aggregation under standard purification conditions.
At a Glance
| Chromatographic Method | Primary Role | Key Condition | Expected Outcome |
|---|---|---|---|
| Anion Exchange (Q or DEAE) | Capture and initial enrichment | pH 7.5 to 8.5 buffer, salt gradient elution | Removes most host cell proteins and nucleic acids |
| Size Exclusion Chromatography | Final polishing and monomer isolation | Isocratic elution in physiological buffer | Separates monomer from oligomers and aggregates |
| Affinity Tag Purification | Optional alternative for recombinant protein | Immobilized metal or antibody resin | Single-step capture but requires tag removal |
Understanding Alpha-Synuclein's Purification Challenge
Alpha-synuclein is a 140 amino acid protein that lacks a stable three dimensional structure under native conditions. This intrinsically disordered character creates a distinctive purification problem because the protein does not fold into a compact globular shape that most chromatographic resins are designed to handle. The disordered state means that alpha-synuclein behaves differently on columns compared to well folded proteins, and it is particularly sensitive to conditions that promote misfolding and aggregation.
The protein's primary sequence contains three regions that influence purification behavior. The N terminal region contains conserved repeats that are involved in membrane binding. The central region, known as the NAC domain, is highly hydrophobic and drives the aggregation process. The C terminal region is acidic and contains the majority of the charged residues that determine the protein's chromatographic behavior. Understanding these regions is essential because each one affects how the protein interacts with different resins and buffers.
The aggregation propensity of alpha-synuclein is the central challenge in its purification. The hydrophobic NAC domain promotes self association, and this process is accelerated by elevated temperature, high protein concentration, and the presence of certain metal ions. Aggregation is not simply a nuisance that reduces yield. It also creates a safety concern because the resulting fibrils are the pathological species associated with Parkinson disease and other synucleinopathies. Purification protocols must therefore be designed to minimize the time that the protein spends in conditions that favor aggregation.
The choice of expression system also affects the purification strategy. Recombinant alpha-synuclein is commonly expressed in bacterial systems because the protein is small and does not require post translational modifications. However, bacterial expression introduces host cell proteins and nucleic acids that must be removed during purification. The purification strategy must account for these contaminants while also protecting the protein from aggregation. The Addgene protocols provide general guidance on expression and lysis workflows that can be adapted for alpha-synuclein purification.
Core Principles of Alpha-Synuclein Chromatography
The Intrinsically Disordered State and Its Implications
The disordered nature of alpha-synuclein means that it does not have a well defined hydrophobic core that would normally drive binding to reversed phase resins. This property makes reversed phase chromatography a poor choice for the initial purification because the protein may not bind predictably and may be exposed to organic solvents that promote aggregation. Instead, the charged residues in the C terminal region provide the basis for ion exchange chromatography, which is the most reliable first step.
The C terminal region of alpha-synuclein contains multiple acidic residues that give the protein a low isoelectric point. This means that at neutral pH, the protein carries a net negative charge and will bind to anion exchange resins. The binding is relatively weak compared to many other proteins because the charge is distributed across a disordered region instead of concentrated in a structured domain. This weak binding is actually an advantage because it allows the protein to be eluted with a gentle salt gradient that does not expose it to harsh conditions.
The disordered structure also affects how the protein interacts with size exclusion chromatography. Because alpha-synuclein does not fold into a compact globular shape, it behaves as a larger molecule than its molecular weight would suggest. The protein appears to have a hydrodynamic radius that is larger than expected for a 14 kDa protein, which means it elutes earlier than a globular protein of the same mass. This behavior must be accounted for when interpreting size exclusion chromatograms and when calibrating the column with molecular weight standards.
Temperature and Buffer Conditions
Temperature is the most critical variable in alpha-synuclein purification. The protein aggregates more rapidly at elevated temperatures, and even brief exposure to room temperature can reduce the yield of monomeric protein. Purification should be performed at cold temperatures whenever possible, and the protein should be kept on ice between steps. The cold room is the preferred environment for the entire purification procedure.
Buffer composition also plays a critical role in maintaining the monomeric state. The pH should be maintained in the range of 7.0 to 8.5 to keep the protein in its native disordered state. The buffer should contain a reducing agent such as dithiothreitol or tris(2-carboxyethyl)phosphine to prevent oxidation of the protein's methionine residues. Oxidation can promote aggregation and should be minimized throughout the purification.
The ionic strength of the buffer is a balance between maintaining protein solubility and allowing binding to the ion exchange resin. The protein is more soluble at moderate salt concentrations, but the binding to the anion exchange resin requires low salt conditions. The typical approach is to load the protein in a low salt buffer and then elute with a gradient to higher salt. The salt concentration should be increased gradually to avoid local concentration effects that could promote aggregation.
Practical Workflow for Purification
Step 1: Preparation of the Cell Lysate
The purification begins with the preparation of a cleared lysate from the expression host. The cells should be resuspended in a lysis buffer that contains a protease inhibitor cocktail to prevent degradation of the protein. The lysis buffer should also contain a reducing agent and a chelating agent such as ethylenediaminetetraacetic acid to remove metal ions that could promote aggregation.
The cells are typically lysed by sonication or high pressure homogenization. The lysis method should be chosen based on the scale of the preparation and the equipment available in the laboratory. Sonication is suitable for small volumes, while high pressure homogenization is more appropriate for larger cultures. The lysate should be kept cold throughout the lysis process to minimize protein degradation and aggregation.
The lysate is then clarified by centrifugation to remove cell debris and insoluble material. The centrifugation should be performed at high speed and the supernatant should be carefully collected without disturbing the pellet. The clarified lysate can be further filtered through a membrane to remove any remaining particulate material that could clog the chromatography column.
Step 2: Anion Exchange Chromatography
The clarified lysate is loaded onto an anion exchange column that has been equilibrated with the starting buffer. The column should be a strong anion exchanger such as a quaternary ammonium resin to ensure complete binding of the protein. The loading should be performed at a low flow rate to allow the protein to bind efficiently to the resin.
After loading, the column is washed with the starting buffer to remove unbound proteins and other contaminants. The wash step is important because it removes the majority of host cell proteins that do not bind to the resin. The wash should be continued until the absorbance at 280 nm returns to the baseline.
The protein is then eluted with a linear salt gradient. The gradient should be shallow to allow the protein to separate from other bound proteins. The alpha-synuclein typically elutes at a moderate salt concentration, and the elution should be monitored by absorbance at 280 nm. The fractions containing the protein should be pooled based on the chromatogram and the results of the analysis.
Step 3: Size Exclusion Chromatography
The pooled fractions from the anion exchange step are concentrated and then applied to a size exclusion column. The size exclusion column should be selected based on the molecular weight range of the protein and the expected contaminants. The column should be equilibrated with a physiological buffer that is compatible with the downstream applications.
The size exclusion step serves two purposes. It removes any remaining high molecular weight contaminants, and it separates the monomeric alpha-synuclein from any oligomers or aggregates that may have formed during the earlier steps. The monomeric protein should elute as a single peak, and the fractions containing the monomer should be collected.
The size exclusion step also provides an opportunity to exchange the protein into a final buffer that is suitable for storage or downstream use. The protein can be eluted directly into the desired buffer, avoiding the need for a separate dialysis step. The final protein should be analyzed to confirm its purity and its monomeric state.
Options and Tradeoffs in Purification Strategy
Anion Exchange Versus Affinity Purification
The choice between anion exchange and affinity purification depends on the expression system and the intended use of the protein. Anion exchange is a general method that does not require any specific tag on the protein. It is suitable for purifying both recombinant and native alpha-synuclein, and it does not require the removal of a tag after purification.
Affinity purification requires the protein to be expressed with a tag such as a histidine tag or a glutathione S transferase tag. The tag allows the protein to be purified in a single step using a resin that binds the tag. This approach is faster and can be more convenient, but it requires the tag to be removed after purification if the protein is to be used for structural or functional studies. The tag removal step adds time and can introduce additional aggregation risk.
The choice between the two approaches depends on the downstream application. If the protein is to be used for structural studies, the tag should be removed and the protein should be purified by anion exchange and size exclusion. If the protein is to be used for routine assays, the tag may be retained and the affinity purification may be sufficient.
The Role of Size Exclusion in the Purification
Size exclusion chromatography is an essential step in the purification of alpha-synuclein because it is the only method that can reliably separate the monomer from the aggregated species. The aggregation of alpha-synuclein is a continuous process, and even the best purification will contain some oligomeric species. The size exclusion step removes these species and ensures that the final protein is predominantly monomeric.
The size exclusion step also provides a measure of the quality of the purification. The chromatogram should show a single peak for the monomeric protein. If the chromatogram shows multiple peaks or a shoulder on the monomer peak, this indicates that the protein has aggregated during the purification. The presence of aggregates should be investigated and the purification conditions should be adjusted to minimize the aggregation.
The size exclusion step can also be used to determine the apparent molecular weight of the protein. The elution volume of the protein can be compared to the elution volumes of molecular weight standards to estimate the size of the protein. This estimate is not accurate for disordered proteins because they do not behave like globular proteins, but it can provide a useful indication of the protein's state.
Observations and Measurements
Monitoring the Purification
The purification should be monitored at each step to ensure that the protein is being purified correctly and that the protein is not being lost or degraded. The most common method for monitoring the purification is the measurement of the absorbance at 280 nm. The absorbance is used to detect the protein in the column eluate and to determine the concentration of the protein in the fractions.
The purity of the protein should be assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The gel should show a single band at the expected molecular weight of the protein. The gel should also be stained to detect any contaminating proteins. The purity of the protein should be at least 95 percent for most applications.
The identity of the protein should be confirmed by mass spectrometry or by immunoblotting with an antibody specific for alpha-synuclein. The mass spectrometry can provide the exact molecular weight of the protein and can confirm that the protein has not been degraded or modified. The immunoblotting can confirm the identity of the protein and can detect any degradation products.
Quantification of the Protein
The concentration of the purified protein should be determined by a method that is appropriate for the protein. The absorbance at 280 nm can be used to determine the concentration if the extinction coefficient of the protein is known. The extinction coefficient can be calculated from the amino acid sequence of the protein, and the concentration can be calculated using the Beer-Lambert law.
The Bradford assay is a common method for protein quantification, but it is not reliable for alpha-synuclein because the protein is intrinsically disordered and does not bind the dye in a predictable manner. The BCA assay is also commonly used, but it can be affected by the presence of reducing agents in the buffer. The choice of the quantification method should be validated for the specific buffer and the protein.
The concentration of the protein should be measured at the end of the purification and before the protein is used for any downstream application. The concentration should be recorded in the laboratory notebook along with the other details of the purification. The concentration is important for the interpretation of the downstream experiments.
Records and Quality Control
Documentation of the Purification
The purification should be documented in the laboratory notebook with the details of each step. The documentation should include the date of the purification, the expression culture used, the lysis method, the column type, the buffer composition, the flow rate, and the gradient. The documentation should also include the chromatograms and the gel images.
The documentation is essential for the reproducibility of the purification. The purification should be reproducible, and the documentation should allow another researcher to repeat the purification. The documentation should also be used to troubleshoot any problems that occur during the purification.
The documentation should be stored in a secure location and should be available for review by the laboratory supervisor or the institutional biosafety committee. The documentation should be kept for the duration of the project and should be archived according to the institutional policy.
Common Failure Patterns
The most common failure in the purification of alpha-synuclein is the aggregation of the protein. The aggregation can be detected by the presence of a precipitate in the protein solution, by the presence of multiple peaks in the size exclusion chromatogram, or by the presence of high molecular weight bands on the SDS-PAGE gel. The aggregation is usually caused by the protein being exposed to conditions that favor the aggregation, such as elevated temperature, high protein concentration, or the presence of metal ions.
The aggregation can be minimized by keeping the protein cold, by working quickly, and by avoiding the conditions that promote aggregation. The protein should be kept at a concentration below the threshold for aggregation, and the buffer should contain a reducing agent and a chelating agent. If the protein aggregates, the purification should be repeated with the adjusted conditions.
Another common failure is the loss of the protein during the purification. The protein can be lost by binding to the column or by precipitation during the concentration step. The loss can be minimized by using the appropriate column and by the protein being concentrated gently. The loss should be monitored by the measurement of the protein concentration at each step.
Biosafety and Regulatory Context
Biosafety Considerations
The purification of alpha-synuclein is generally performed under biosafety level 1 conditions, but the specific requirements depend on the expression system and the nature of the protein. The Biosafety in Microbiological and Biomedical Laboratories provides the principles for the risk assessment and the containment of the biological materials. The laboratory should conduct a risk assessment before the purification and should follow the institutional biosafety requirements.
The NIH Guidelines for Recombinant or Synthetic Nucleic Acid Molecules provide the official oversight for the research involving the recombinant nucleic acids. The purification of the recombinant alpha-synuclein is subject to these guidelines, and the laboratory should follow the appropriate containment and the oversight requirements.
The Safety of the Protein
Alpha-synuclein is not considered a hazardous material, but the protein can be a hazard if it is inhaled or if it comes into contact with the skin. The protein should be handled with the appropriate personal protective equipment, including the laboratory coat, the gloves, and the eye protection. The protein should be handled in a biosafety cabinet if the protein is being used in a form that could generate aerosols.
The protein should be disposed of in accordance with the institutional requirements for the disposal of the biological materials. The protein should be inactivated before disposal, and the disposal should be documented. The protein should not be released into the environment.
The Role of Standards and Quality
The Importance of Measurement Standards
The quality of the purified alpha-synuclein should be assessed using the appropriate measurement standards. The NIST Laboratory Programs provide the measurement science and the standards that can be used to ensure the quality of the measurements. The laboratory should use the standards that are appropriate for the specific measurement and the protein.
The measurement of the protein concentration should be validated using a standard curve that is prepared with a known protein standard. The standard curve should be prepared with the same buffer as the protein, and the standard should be measured in the same manner as the protein. The standard curve should be used to calculate the concentration of the protein.
The measurement of the purity should be validated using a standard protein that is known to be pure. The standard protein should be run on the same gel as the protein, and the purity of the protein should be compared to the purity of the standard. The purity of the protein should be at least as high as the purity of the standard.
The Use of Controls
The purification should include the appropriate controls to ensure the quality of the purification. The controls should include a negative control that is a sample that does not contain the protein, and a positive control that is a sample that is known to contain the protein. The controls should be processed in the same manner as the protein, and the results should be compared to the results of the protein.
The controls should be used to validate the purification and to ensure that the results are not due to the contamination or the artifacts. The controls should be included in the documentation and should be used to interpret the results of the purification.
The Interpretation of the Purification Results
The Analysis of the Purification
The results of the purification should be analyzed to determine the yield and the purity of the protein. The yield is the amount of the protein that is recovered at the end of the purification, and the purity is the fraction of the protein that is the target protein. The yield and the purity should be calculated and recorded.
The yield is calculated by dividing the amount of the protein at the end of the purification by the amount of the protein at the beginning of the purification. The yield should be expressed as a percentage, and the yield should be at least 50 percent for a successful purification. The purity should be calculated by dividing the amount of the target protein by the total amount of the protein in the sample.
The yield and the purity should be used to evaluate the success of the purification and to identify the areas for the improvement. The yield and the purity should be compared to the expected values, and the differences should be investigated.
The Interpretation of the Results
The results of the purification should be interpreted in the context of the downstream application. The protein should be pure enough and the protein should be in the correct state for the downstream application. The protein should be monomeric and the protein should be in the correct buffer.
The results should be interpreted in the context of the limitations of the purification. The purification is not a guarantee of the quality of the protein, and the protein should be validated for the downstream application. The protein should be tested for the activity and the protein should be tested for the aggregation.
The results should be documented and the results should be used to plan the future experiments. The results should be shared with the laboratory and the results should be used to improve the purification.
The Role of the Purification in the Research
The Use of the Purified Protein
The purified alpha-synuclein is used for a variety of the downstream applications, including the structural studies, the biophysical studies, and the functional studies. The protein is used to study the structure of the protein and the protein is used to study the aggregation of the protein. The protein is used to study the function of the protein and the protein is used to study the interaction of the protein with the other molecules.
The purified protein is also used for the development of the diagnostic and the therapeutic agents. The protein is used to screen for the compounds that can inhibit the aggregation of the protein, and the protein is used to develop the antibodies that can detect the protein. The protein is used to develop the models of the disease and the protein is used to test the potential of the treatments.
The Limitations of the Purification
The purification of the alpha-synuclein has the limitations that should be considered. The purification is a time-consuming and the purification is a labor-intensive process. The purification requires the specialized equipment and the purification requires the specialized skills. The purification is not a guarantee of the quality of the protein, and the protein should be validated for the downstream application.
The purification is also limited by the intrinsic properties of the protein. The protein is prone to the aggregation, and the protein is prone to the degradation. The purification should be designed to minimize the aggregation and the degradation, but the purification cannot completely eliminate the risk. The protein should be used as soon as possible after the purification, and the protein should be stored in the appropriate conditions.
The Future of the Purification
The Advances in the Purification
The purification of the alpha-synuclein is an active area of the research, and the advances are being made in the purification methods. The advances include the development of the new resins and the development of the new buffers. The advances include the development of the new methods for the detection of the protein and the development of the new methods for the quantification of the protein.
The advances are being made in the understanding of the protein and the advances are being made in the understanding of the aggregation. The advances are being made in the development of the new tools for the purification and the advances are being made in the development of the new methods for the analysis of the protein.
The advances are being made in the development of the new methods for the purification of the protein and the advances are being made in the development of the new methods for the use of the protein. The advances are being made in the development of the new methods for the study of the protein and the advances are being made in the development of the new methods for the treatment of the disease.
A Decision Framework for Choosing Between Anion Exchange and Affinity Capture
The choice between anion exchange and affinity capture is not a one time decision that applies to every preparation. The correct choice depends on the expression construct, the downstream application, the available equipment, and the scale of the preparation. This section provides a practical decision framework that a laboratory can apply before starting a purification. The framework uses a series of questions that lead to a specific capture strategy, and it includes a record system for tracking the decisions and their outcomes.
Step 1: Define the Downstream Application
The first question is what the purified protein will be used for. Structural studies such as nuclear magnetic resonance spectroscopy or X ray crystallography require tag free protein at high concentration and high purity. Biophysical studies such as circular dichroism or dynamic light scattering also require tag free protein because the tag can alter the aggregation behavior. Functional assays such as membrane binding or lipid vesicle experiments may tolerate a tag, but the tag should be validated to confirm that it does not change the activity.
If the downstream application requires tag free protein, the affinity capture approach adds a protease cleavage step and a second purification to remove the tag and the protease. Each additional step increases the time that the protein spends in solution and increases the risk of aggregation. In this case, anion exchange as the capture step is usually the better choice because it does not require a tag and it produces protein that is ready for the size exclusion step.
If the downstream application tolerates a tag, the affinity capture approach can be faster and can produce protein in fewer steps. The tag also provides a convenient handle for detection and for pull down experiments. The decision should be recorded in the laboratory notebook with the rationale for the choice.
Step 2: Assess the Expression Construct
The expression construct determines whether affinity purification is even possible. A histidine tag or a glutathione S transferase tag must be present in the construct for affinity purification to work. If the construct does not have a tag, anion exchange is the only option for the capture step. If the construct has a tag, the decision depends on the downstream application and on the expected behavior of the tag.
The histidine tag is the most common tag for alpha-synuclein because it is small and does not add much mass to the protein. The histidine tag binds to immobilized metal resins such as nickel or cobalt. The binding is strong and the elution is achieved with imidazole. The glutathione tag is larger and can affect the behavior of the protein on the size exclusion column. The glutathione tag also requires a specific resin and a specific elution condition.
The tag should be tested for its effect on the aggregation behavior of the protein. Some tags can stabilize the protein and reduce aggregation, while others can promote aggregation. The effect of the tag should be assessed by comparing the aggregation behavior of the tagged and the untagged protein. This comparison should be performed before the full purification is attempted.
Step 3: Evaluate the Scale of the Preparation
The scale of the preparation affects the choice of the capture step. For small scale preparations, such as a 1 liter culture, the affinity purification is convenient because it can be performed in a single day and does not require a large column. For large scale preparations, such as a 10 liter culture, the anion exchange is often more practical because the resin is less expensive and the column can be scaled up more easily.
The scale also affects the choice of the lysis method. Sonication is suitable for small volumes but is not practical for large volumes. High pressure homogenization is the preferred method for large volumes because it is faster and more reproducible. The lysis method should be chosen based on the scale and the equipment available in the laboratory.
The scale should be recorded in the laboratory notebook along with the culture volume and the expected yield. The expected yield should be estimated from the expression level and the purification method. The actual yield should be compared to the expected yield to identify any problems.
Step 4: Consider the Equipment and the Expertise
The equipment available in the laboratory affects the choice of the purification method. Anion exchange requires a chromatography system that can run a salt gradient. Affinity purification can be done with a simple gravity column or with a chromatography system. The size exclusion step requires a chromatography system with a fraction collector.
The expertise of the person performing the purification also affects the choice. Anion exchange is a more general method that requires more experience to optimize. Affinity purification is more straightforward and can be performed by a person with less experience. The choice should be made based on the expertise of the person and the time available for the purification.
The equipment and the expertise should be recorded in the laboratory notebook with the details of the purification. The record should include the type of the chromatography system, the column type, and the person who performed the purification.
Step 5: Record the Decision and the Outcome
The decision should be recorded in the laboratory notebook with the date, the rationale, and the expected outcome. The outcome should be recorded after the purification is complete. The outcome should include the yield, the purity, and the state of the protein. The outcome should be compared to the expected outcome and the differences should be investigated.
The record should be used to improve the purification over time. The record should be reviewed after each purification and the decision should be adjusted based on the outcome. The record should be shared with the laboratory and the record should be used to plan the future purifications.
The record should also include the details of the buffer and the column. The buffer composition and the column type should be recorded for each purification. The record should include the flow rate and the gradient. The record should include the chromatogram and the gel image.
A Comparison of the Two Capture Strategies
| Criterion | Anion Exchange | Affinity Purification |
|---|---|---|
| Tag requirement | None | Required |
| Number of steps | Two (anion exchange and size exclusion) | Two or three (affinity, tag removal, size exclusion) |
| Time required | Longer | Shorter |
| Cost per preparation | Lower | Higher |
| Suitability for tag free protein | Yes | No |
| Suitability for large scale | Yes | Less suitable |
| Expertise required | More | Less |
| Risk of aggregation | Moderate | Moderate to higher |
The table provides a summary of the tradeoffs between the two capture paths. The choice should be made based on the specific requirements of the downstream application and the available resources. The table should be used as a starting point for the decision and the decision should be adjusted based on the specific circumstances.
The Role of the Size Exclusion Step in Both Paths
The size exclusion step is required in both paths because it is the only method that can reliably separate the monomer from the aggregated species. The size exclusion step should be performed after the capture step and before the final analysis. The size exclusion step also provides a way to exchange the protein into the final buffer.
The size exclusion step should be performed with a column that has a suitable fractionation range. The column should be calibrated with the molecular weight standards to determine the elution volume of the monomer. The monomer should elute as a single peak and the fractions containing the monomer should be collected.
The size exclusion step should be monitored by the absorbance at 280 nm. The chromatogram should show a single peak for the monomer. If the chromatogram shows multiple peaks or a shoulder, the protein has aggregated and the purification should be repeated with the adjusted conditions.
The Use of the Decision Framework in the Laboratory
The decision framework should be used in the laboratory to make the purification decisions. The framework should be applied to each purification and the decision should be recorded. The framework should be used to improve the purification over time and the framework should be used to train the new members of the laboratory.
The framework is a practical tool that can be used to make the purification more efficient and more reproducible. The framework should be used in combination with the other sections of this article and the framework should be adjusted based on the specific circumstances.
The framework should be documented in the laboratory notebook and the framework should be available for the review by the laboratory supervisor. The framework should be used to ensure that the purification is performed in a consistent manner and the framework should be used to identify the areas for the improvement.
Frequently Asked Questions
Why is alpha-synuclein difficult to purify?
Alpha-synuclein is intrinsically disordered and does not have a stable three-dimensional structure. This means it does not behave like a typical globular protein on chromatography columns, and it is prone to aggregation under conditions that would be harmless to most proteins. The hydrophobic NAC domain promotes self association, and the protein is sensitive to temperature, concentration, and buffer conditions.
What is the best first step for purifying alpha-synuclein?
Anion exchange chromatography is the most reliable first step because the C terminal region of the protein carries a net negative charge at neutral pH. The protein binds to a strong anion exchange resin and is eluted with a salt gradient. This step removes the majority of host cell proteins and nucleic acids from the lysate.
Why is size exclusion chromatography necessary?
Size exclusion chromatography is necessary to separate the monomeric alpha-synuclein from the oligomers and aggregates that form during the purification. The protein is prone to aggregation, and the size exclusion step is the only method that can reliably remove the aggregated species. The size exclusion step also provides a way to exchange the protein into a final buffer.
Should I use a His tag for purification?
A His tag can be used for a single step purification, but the tag must be removed if the protein is intended for structural or functional studies. The tag removal step adds time and can introduce aggregation risk. Anion exchange and size exclusion chromatography can produce tag free protein without the need for a tag.
How do I prevent the aggregation of the protein?
The aggregation can be minimized by keeping the protein cold, working quickly, and avoiding the conditions that promote the aggregation. The buffer should contain a reducing agent and a chelator, and the protein should be kept at a concentration below the threshold for the aggregation. The protein should be stored in the appropriate conditions and should be used as soon as possible.
How do I measure the purity of the protein?
The purity of the protein is measured by SDS-PAGE and the gel is stained to detect the protein. The purity should be at least 95 percent for most applications. The identity of the protein should be confirmed by mass spectrometry or by western blotting with an antibody specific for alpha-synuclein.
What is the yield of a typical purification?
The yield of the purification depends on the expression level and the purification method. The yield should be at least 50 percent for a successful purification. The yield should be calculated by dividing the amount of the protein at the end of the purification by the amount of the protein at the beginning of the purification.
What are the biosafety considerations for the purification?
The purification of the recombinant alpha-synuclein should be performed under the biosafety level 1 conditions. The laboratory should conduct a risk assessment and should follow the institutional biosafety requirements. The Biosafety in Microbiological and Biomedical Laboratories provides the principles for the biosafety and the NIH Guidelines for Recombinant or Synthetic Nucleic Acid Molecules provide the official oversight for the research.
Related Molecular Biology Guides
- Protein Expression and Purification: A Practical Guide
- Custom Protein Purification: Methods, Mechanisms, and Pitfalls
- His Tag Protein Purification: Principles and Protocols
- His-Tag Purification Resin: Principles and Protocols
- His-Tagged Protein Purification: Principles and Methods
References and Further Reading
- Molecular Biology and Laboratory Methods. National Library of Medicine.
- Biosafety in Microbiological and Biomedical Laboratories. CDC and NIH.
- NIH Guidelines for Recombinant or Synthetic Nucleic Acid Molecules. National Institutes of Health.
- Addgene Protocols. Addgene.
- MIQE Guidelines. Clinical Chemistry.
- NIST Laboratory Programs. National Institute of Standards and Technology.
- Expression and Purification of Untagged α-Synuclein.. Methods in molecular biology (Clifton, N.J.), 2019.
- Purification of Recombinant α-synuclein: A Comparison of Commonly Used Protocols.. Biochemistry, 2020.
- Recombinant Expression and Purification of N-Acetylated Alpha-Synuclein.. Methods in molecular biology (Clifton, N.J.), 2019.
- Purification of α-synuclein from human brain reveals an instability of endogenous multimers as the protein approaches purity.. Biochemistry, 2015.
- A method of purifying alpha-synuclein in E. coli without chromatography.. Heliyon, 2021.
This article is educational and does not replace validated laboratory procedures, institutional biosafety review, manufacturer instructions, or professional interpretation.