Purifying the protein you want to study is one of the most critical research steps. Protein purification entails four fundamental procedures:
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Cell lysis
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Binding of proteins to a matrix
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Washing
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Elution
Various techniques can prove instrumental during cell lysis, such as non-enzymatic approaches, the use of hydrolytic enzymes like lysozyme, or the application of detergent reagents. As natural proteins are notoriously difficult to isolate, recombinant proteins of interest also require specific processing for successful purification and isolation.
This protein guide provides a general overview of the protein purification method, including tips on choosing the appropriate technique to purify protein.
Why is protein purification essential?
Proteins are essential for almost every physiological action, including transcription, translation, replication, metabolism, signaling between cells, and even apoptosis. DNA contains the instructions for making proteins and is used as a blueprint by transcriptional processes to generate messenger RNA (mRNA). Eventually, the information contained in mRNA is translated into the ordered chains of amino acids that make up a protein. All organisms produce proteins using a similar two-step process: the transcription of DNA into RNA and the translation of RNA into proteins.
Recombinant Proteins: What are they?
Recombinant proteins are those that have been created by cloning recombinant DNA into an expression vector that allows the gene to be expressed and messenger RNA to be translated. The use of recombinant DNA technology to modify a gene can result in the production of a mutant protein. A recombinant protein is an engineered version of a naturally occurring protein that can be used to boost output, modify gene sequences, and create breakthrough consumer products at a commercial level.
Why is protein purification so essential?
The host cell is responsible for the production of recombinant proteins as well as a wide range of other cellular compounds. Consequently, for most applications, such as therapeutics, recombinant proteins require extraction and purification from the remaining cellular molecules.
The purification process can vary significantly depending on the type of protein. For instance, membrane proteins are challenging to purify since they are often produced in small quantities and require detergents to solubilize in aqueous solutions. Furthermore, when purifying membrane proteins, it is vital to choose detergents that can effectively dissolve and stabilize the specific membrane protein.
Evolution of Protein Purification Methods
It has long been understood that separating proteins in biological samples is a crucial and challenging task with far-reaching consequences for human health. Along with immunoassays, affinity extraction methods, electrophoresis, and chromatography, current approaches to protein separation have roots in early separation technologies developed in the 1950s and 1960s. Today, it is necessary to separate and identify numerous low-abundance proteins inside complex biological matrices.
Understanding the fundamentals of protein separation from a molecular and nanoscale perspective will allow today’s scientists to design future purification solutions.
Protein Purification Strategies
Purifying proteins is a fundamental prerequisite for studying them individually or in complexes, as well as determining how they interact with other molecules such as nucleic acids. Protein purification methods can be scaled up or down depending on the amount of protein to be extracted and the intended downstream applications. In many cases, the optimal strategy is determined through empirical testing to see what works best.
So, what are the most effective purification methods when working with proteins?
The Right Purification Method for the Right Protein
The most effective protein purification technique depends on the protein of interest, the host cell utilized to generate it, and several other parameters. Because of its versatility, rapid growth, and low cost, Escherichia coli (E. coli) has always been the preferred organism for many researchers for producing recombinant proteins. Proteins made in E. coli can be produced in large quantities; however, some of them, especially eukaryotic proteins, may fail to function or fold correctly.
Mammalian cell culture presents a promising alternative for efficiently producing fully folded and functional mammalian proteins, complete with the necessary post-translational modifications. However, the difficulty in purifying these recombinant proteins stems from their low expression levels in cultivated mammalian cells. This means that the highly selective and efficient capture of these proteins from bulk cell lysates is essential for achieving sufficient yield and purity.
Recombinant proteins of interest can have affinity purification tags added to them to facilitate purification. Enzymes, small proteins, and polypeptides often serve as fusion tags and are added to a recombinant protein's N- or C-terminus. Certain tagged proteins may exhibit changes in their stability, solubility, and expression depending on the tag's biochemical properties. Expression vectors that incorporate fusion tags make isolating recombinant proteins easier.
Chromatography in Protein Purification
Chromatography, a collection of various techniques, is the foundation of most processes used to purify recombinant proteins. Chromatography methods separate molecules based on similarities and differences between the target protein and the rest of the sample. These qualities can include molecular structure, surface charge, and size.
Affinity chromatography is the most common way to purify proteins. This method separates proteins based on their specific interaction with a matrix. It is one of the most effective strategies because it incorporates a desired structure (known as a tag) onto the protein. This tag is unique to the target protein in the sample and provides specific properties that help isolate it from the remaining molecules. However, in cases where a molecule cannot be tagged, researchers must resort to alternative techniques.
Protein Complex Isolation
The elucidation of protein function and the structure of the complex systems responsible for essential biological activities is a fundamental goal of proteomics. Insight into the cell signaling cascades that underlie these activities is achievable through the investigation of protein complexes.
For instance, transcription factors play a significant role in regulating the transcription process by binding to specific recognition sites on DNA, most frequently at the gene promoter, and interacting with other proteins within the nucleus. Cell survival, differentiation, and expansion depend on this regulation.
Protein Analysis
Stable methods for immobilizing proteins on solid surfaces in specific orientations without affecting protein structure or function are often required to study protein interactions. Affinity tags are a valuable tool for this immobilization since they rarely affect binding efficiency. Protein immobilization on chips is a standard method for protein analysis.
DNA and Amino Acids
Typically, functional protein microarrays comprise active proteins or protein domains attached to a solid surface along their entire length. Probes made of fluorescently labeled DNA are used to screen the array and find the proteins that bind to each probe. Protein microarrays provide a high-throughput approach for characterizing protein-DNA interactions.
Protein Identification
Protein binding partners can be isolated in vivo or in vitro using immobilized proteins in pull-down assays. Other downstream applications, such as mass spectrometry, do not require protein immobilization to identify protein partners and individual components of protein complexes.
Affinity Tags for Purifying Proteins
Affinity tags are used to isolate or immobilize a target protein. These tags come in a variety of forms; therefore, they help scientists achieve successful purification across different protein classes and meet specific purification criteria.
Glutathione-S-Transferase (GST)
Glutathione-S-transferase (GST) is used as an affinity tag because of its high specificity for immobilized, glutathione-coated matrices. Many eukaryotic species possess glutathione-S-transferases, which belong to a family of multifunctional cytosolic proteins. Because GST isoforms are typically absent in most bacteria, endogenous bacterial proteins do not compete with GST-fusion proteins for binding to the purification resin. The 26 kDa GST affinity tag also augments the solubility of numerous bacterially produced eukaryotic proteins.
Polyhistidine (His-tag)
The polyhistidine tag stands as the most widely employed of all tags. Its capacity to purify proteins depends on the attraction of histidine residues to immobilized metal ions, such as nickel. The interaction is driven by the coordination of the nitrogen atoms in the imidazole rings of the histidine residues with a free binding spot on the metal. The metal ion is firmly bound to the solid support through a chelating agent covalently bonded to the resin.
Using these tags has various benefits for protein purification. Because of its small size, the polyhistidine tag is less immunogenic than larger tags. As a result, the tag does not typically require removal for downstream applications after the purification process is complete. The target protein can be tagged at either the C-terminus or the N-terminus. Lastly, unlike many other tags, the polyhistidine tag's affinity for the metal is not dependent on the secondary structure of the tag, allowing the use of denaturing conditions to help purify insoluble proteins.
Purifying of Polyhistidine-Tagged Proteins
Magnetic Resins for Purifying Polyhistidine-Tagged Proteins
There is a rising demand for high-throughput screening-compatible protein purification techniques. Magnetic resins allow for the purification of affinity-tagged proteins without requiring additional centrifugation processes or sample transfers between tubes. A successful purification resin must meet several requirements, including minimal nonspecific protein binding, a strong affinity for the target protein, and effective fusion protein recovery.
The MagneGST™ system for purifying proteins meets these criteria, making it a viable option for protein purification across a broad range of expression levels and molecular weights. The magnetic characteristics of the binding materials enable the performance of purification within a single tube, even if the process begins with a crude lysate. The technology is also compatible with high-throughput automated liquid-handling units. To create effective frameworks for automated systems, it is recommended to consult the manufacturer's manuals for insight.
Purification of Bacterial-Cell-Expressed Proteins
Recombinant proteins expressed in bacterial cells may be sequestered in inclusion bodies. Use FastBreak™ Cell Lysis Reagent (10X) to lyse your cells and determine if the protein is located in an inclusion body. Separate the cell debris by centrifugation into a pellet, then use gel analysis to examine the pellet and supernatant for polyhistidine-tagged proteins.
Denaturing conditions are necessary for the effective purification of insoluble proteins. Since the interaction between polyhistidine-tagged fusion proteins and MagneHis™ Ni-Particles is independent of tertiary structure, fusion proteins can be collected and purified under denaturing conditions by introducing a potent denaturant, such as 2–8 M guanidine hydrochloride or urea, to the cells.
To prevent the proteins from forming larger aggregates, denaturing conditions must be maintained throughout the procedure. MagneHis™ Binding/Wash and Elution Buffers should be directly mixed with solid guanidine-HCl or urea to create denaturing buffers.
Purification from Insect and Mammalian Cells
Cells should be processed at a density of 2 × 106 cells/mL. Cells that have adhered to the tissue culture vessel can be detached using a scraper and resuspended in culture media at this concentration. Processing of cells can take place in a culture medium that contains up to 10% serum. Processing a higher concentration of cells per milliliter of material could lead to a reduction in target protein yield and a rise in nonspecific binding. To purify proteins secreted into the cell culture medium, it is not necessary to lyse the cells (unless the protein is also retained intracellularly).
Other Popular Purification Techniques
Size-Exclusion Chromatography (Gel Filtration)
Gel filtration, also known as size-exclusion chromatography (SEC), is a technique that uses molecular size to separate substances. Using a resin with pores of a specified width makes it possible to separate molecules based on their ability to travel through the matrix.
Ion Exchange Chromatography (IEX)
Ion exchange chromatography is another technique that has widespread application. This technique separates molecules according to their net surface charge under specific conditions of pH and ionic strength.
HIC and Reverse-Phase Chromatography
Hydrophobic interaction chromatography (HIC) and reverse-phase chromatography (RPC) are typical techniques for separating proteins based on their hydrophobicity (polarity). These techniques are among the most widely utilized in the industry. The hydrophobicity of the matrix that the purified protein interacts with distinguishes these techniques from one another.
Limitations of Non-Affinity Methods
Since it is common to encounter molecules with comparable sizes and charges, these latter methods are often less selective than affinity chromatography. Consequently, they may not separate and purify target proteins as effectively when used as a single step.
Conclusion
Biotechnology has made it easier and more efficient to produce recombinant proteins for a wide variety of applications. Furthermore, the importance of recombinant proteins in developing diagnostics, bioreagents, and pharmaceutical therapeutics has skyrocketed in recent years. Without them, biotechnology would not be at the forefront of modern science. Additionally, we anticipate further advancements in protein expression services, which will aid in treating numerous diseases as protein synthesis and purification technologies continue to evolve.