A protein purification process that works as planned is remarkable. Whether you're working with a recombinant protein produced by E. coli or attempting to separate a protein from a plant or animal membrane, you'll likely be working with an intricate mixture of proteins, nucleic acids, polysaccharides, and lipids.
Moreover, you'll need to obtain milligram (or microgram) quantities of the target protein, typically with exceptional purity and ideally a high yield. Protein purification can be developed as a sequence of fractionation processes aimed at achieving the following results:
- A high concentration of the target protein in a single fraction.
- The removal of a sizable portion of the impurities.
Given these objectives, protein purification protocols incorporate various techniques and face unique challenges. This post covers the common strategies and hindrances faced during protein purification.
What Does Protein Purification Mean?
In biochemistry, a protein is defined as a long chain of amino acids linked together as a polymer. Proteins play a crucial role in how cells are structured and how they function. However, recognizing and isolating proteins is a prerequisite for studying them.
High-throughput protein purification is a common technique biochemists use to achieve this goal in large-scale production. Proteins are first extracted and then separated according to their molecular weight, solubility, charge, and specific binding affinity. This method assists researchers in analyzing individual proteins and studying protein characterization and interactions.
Protein purification is the process of isolating a single protein from a complicated mixture. Once you isolate a protein, it is easier to analyze its properties, including:
- Size and composition
- Bonding affinity
- Biological functions and activities
- Physical and chemical traits
- Interaction with other proteins
Numerous experimental uses exist for protein purification, most notably in the industrial-scale manufacturing of recombinant and complex proteins. However, purifying proteins in their native state is frequently a complicated process. In situations like these, affinity tags, including polyhistidine (His-tag) and glutathione-S-transferase (GST), help isolate and immobilize the target.
How Protein Purification Works?
A protein must go through several steps of purification before it can be properly isolated. The following is a summary of the general steps involved in the process:
1. Cellular Lysis
Cell lysis is when enzymes (like lysozymes), chemicals (like detergents), or physical techniques (like sonication) are used to break open cells to release proteins. Consequently, the lysate obtained from the cells will include either soluble or insoluble proteins, along with organelles, nucleic acids, cell debris, and membrane fragments.
2. Clarification
The next objective is to isolate only purifiable proteins. The lysate undergoes a clarification process to get rid of all cell debris. The method utilized by service providers accomplishes this goal. Once obtained, you can concentrate the protein extract.
3. Protein Capture
The next step is to capture the protein from the crude extract. This phase is also known as the recovery phase. Affinity chromatography and magnetic bead separation are methods used to separate proteins by attaching the protein molecules to a solid matrix.
4. Elution
Elution refers to using a solvent to remove an adsorbed substance. A proper wash buffer is employed to rinse the column and eliminate all non-specific binding. The quantity and duration of rinses will vary with the column type.
Adjusting the pH of the column allows for the desired protein to be eluted. This causes the charged functional groups of proteins to become neutralized, allowing the proteins to be released. The method employs an elution buffer and chemicals such as imidazole (used for His-tagged proteins) to elute the protein of interest at high concentrations. Scientists can collect sufficient amounts of protein for further study using this method.
Protein Purification Strategies and Methods
Protein purification is usually performed using one of four primary techniques. Let's examine each one in greater depth below:
1. Chromatography
One popular method for collecting the desired protein sample is chromatography, which separates compounds based on differences in their physical and chemical characteristics. Purification by column chromatography is a well-known method that utilizes resin-filled columns to isolate the target protein.
Chromatography methods are based on the concept of a mobile phase and a stationary phase.
The stationary phase refers to the resin within the column, whereas the mobile phase refers to the buffer or solvent. In the stationary phase, the protein (or contaminants) in question attaches to the ligand; during the mobile phase, the impurities are removed, or the target protein is eluted.
The method of high-performance liquid chromatography (HPLC) is widely used in the field of chromatography. Depending on the protein that needs purifying, you could use one of the following types of chromatography:
- Affinity chromatography
- Ion-exchange chromatography (Cation and Anion exchange)
- Size-exclusion (Gel filtration) chromatography
- Hydrophobic interaction chromatography (HIC)
2. Precipitation
Salt precipitation is another type of purification technique. It isn't employed as frequently for final purification as it used to be, since it rarely produces completely pure proteins.
The protein in the solution is essentially "salted out" during this procedure. To induce precipitation, high salt concentrations are introduced, with ammonium sulfate serving as the salt of choice. The minimum amount of ammonium sulfate required to precipitate a given protein varies. After separating the protein from the supernatant, the residual salts are removed using dialysis, buffer exchange, or gel filtration chromatography.
3. Isolation (Physical Separation)
Isolation is a fundamental method for the purification of proteins, where the protein can typically be acquired via the following ways:
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Ultracentrifugation: The centrifugal force allows for the separation of proteins. This is used to distinguish between proteins of different sizes and densities. The leftover supernatant and pellet from this form of centrifugation are often used in subsequent purifying steps.
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Fractionation: This technique depends on variations in protein solubility. Since proteins precipitate in highly salty conditions, you can extract them by adding specific amounts of salt.
4. Immunoblotting
Immunoblotting is an extremely popular method for detecting proteins, occasionally used in conjunction with affinity chromatography. It is a method used extensively for identifying proteins and is both quick and precise.
Protein immunoblotting is commonly referred to as Western blotting. Typically, antibodies play a crucial role in a Western blot test in identifying the target protein. The proteins are separated according to their molecular weight using a technique known as SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis). They are then transferred to a membrane and blocked so that antibodies do not bind non-specifically.
After blocking, the membrane is treated with a primary antibody that recognizes the target protein. Then, it is incubated with a secondary antibody that binds to the first antibody. In most cases, the secondary antibody will be coupled to an enzyme or fluorophore that allows for detection. This means that only the target protein will be visualized. Methods of analyzing proteins in this way also serve as valuable indicators of protein purity and expression.
Protein purification is becoming an essential component of biological research. With the discovery of more proteins, it is becoming increasingly necessary to investigate their molecular composition, functions, and interactions with other biomolecules. Laboratories require high-quality chemicals and apparatus to perform purification procedures effectively.
Challenges of Protein Purification
A significant barrier to efficient protein purification is column packing and preparation, which affects both established chromatography methods and cutting-edge technologies like multi-dimensional and mixed-mode chromatography.
Typically, you must dialyze the samples and stain/destain your gels overnight after preparing and packing each of your columns and pouring the gels. Because of how time-consuming and labor-intensive chromatographic processes can be, many researchers view outsourcing this task as a viable option.
In-house column packing allows for a more customized approach and process efficiency but also necessitates a considerable time commitment and material expense. In a typical packing procedure, there are numerous phases involved. These processes include estimating the slurry concentration required for a specific column bed volume, loading the column, evaluating its performance, cleaning it, unpacking it, and storing the resin afterward.
Due to the intricate nature of this process, several challenges arise. These include column setup, resin availability, equipment accessibility, proper storage, user knowledge and training, column failure, and scaling up column quantity. When larger columns are required, in-house column packing, constrained by space and storage requirements, may quickly become a bottleneck in the laboratory workflow.
Variability in column quality and performance across batches is also possible due to the reliance on the specialized experience required for loose resin packing, slurry and buffer production, and column washing. Employing resins sourced from various vendors introduces an additional level of variability. Inconsistency in the procedure can lead to inaccurate results, rendering it challenging to maintain quality control throughout the project. Furthermore, physically handling resins and cleaning columns increases the risk of contamination.
Pre-packed Columns
Many of the challenges inherent to packing resins in-house are avoidable by purchasing pre-packed chromatography columns instead. Pre-packed columns are consistent with Good Manufacturing Practices (GMP) and are ready to use, much like ELISA kits.
Additionally, they come with standardized instructions and validation paperwork. They can be purchased in various sizes, feature a selection of preinstalled resins, and are compatible with most commercially available chromatography equipment. Pre-packed columns are often preferable to in-house packing because they reduce the possibility of cross-contamination and do not necessitate specialized expertise or additional equipment.
Their production further assures data consistency and dependability, even in regulated settings. Pre-packed columns are a modern innovation that can shorten timeframes, boost output, and accelerate process scaling.
Any chromatographer worth their salt understands that the column is where the "magic" occurs. Therefore, pre-packed columns, which come in various compositions with varying degrees of flow characteristics, specificity, and binding capacity, have largely replaced the practice of packing one's own.
Aggregation of Purified Proteins
It is essential to remove protein aggregates from biopharmaceutical APIs because they can make the APIs more immunogenic. However, aggregation can also occur during downstream processing if unfavorable conditions are selected, even though most aggregates are formed during upstream activities. The risk of aggregate formation at the cellular level grows with rising titers.
1. Aggregation Mechanisms
Two main paths lead to the formation of these protein clusters. When a protein is converted from its native to a denatured state, it can expose hydrophobic components that were previously hidden inside. These components can then attach to other molecules with similar hydrophobic elements, forming aggregates.
On the other hand, native proteins may clump together through interactions between the hydrophobic parts of their outer surfaces. In addition, the term "protein aggregation" can describe a wide variety of molecular behaviors. These behaviors may vary from the formation of dimers to the creation of visible particulates. They are caused by noncovalent interactions, mediated by hydrophobicity, and covalent connections generated by the pairing of interchain thiol groups.
2. Minimizing Aggregation
Strategies used to prevent aggregates from forming during downstream processing mainly involve choosing solution conditions and stationary phases (for chromatography) that hinder aggregation and maintain proteins in their stable states. This is occasionally achieved by adding additives or other excipients.
Antioxidants like ascorbic acid and glutathione have been used recently to prevent the oxidation of proteins, which can lead to the development of aggregation sites like aldehydes on lysine residues; these can then interact to generate covalent Schiff-base aggregates. In addition, several biopharmaceutical and bioreagent producers have recently discovered that adding copper during the cell-culture phase results in a substantial decrease in aggregation downstream, although the mechanism is not completely clear.
Conclusion
There is a large variety of protein purification techniques available. In the industrial sector, biotech companies must produce significant quantities of protein. This is because protein purification and expression services perfect their production processes by the time large-scale production begins, needing to generate as many high-quality products as possible.
Purification of proteins takes place on a more minute (small) scale in research laboratories. This is because protein characterization is of primary interest to labs. With the availability of various protein-specific purification methods, businesses and laboratories can take advantage of the benefits of these methods based on their specific objectives.