Getting expression and purification methods right is crucial to writing high-quality scientific papers, but working out the specific steps can be challenging. This resource provides a list of common expression and purification methods. We have also included external links for additional guides on protein purification, expression, and antibody production.
What is Protein Expression?
Proteins must be extensively purified before being employed in research, industry, or medicinal applications. Some proteins, such as casein, which accounts for 80% of the protein in milk, can be efficiently isolated in large amounts from conveniently accessible sources. However, most proteins are not produced in nature in forms and quantities that are easy to purify. By directing cells to create specific proteins in quantities that can be purified for basic research or commercial and therapeutic applications, genetic engineering approaches overcome the constraints of naturally produced proteins. This is the core objective of a protein expression service.
The Protein Production Process
In a protein production service, proteins are generated and regulated in response to a cell's functional requirements. The DNA that contains the blueprints for proteins serves as a template for highly controlled transcriptional mechanisms that result in messenger RNA (mRNA). The message within an mRNA transcript is then translated into a sequence of amino acids that make up a protein. Transcription is the process by which information is copied from DNA to mRNA, while translation is the process by which a protein is synthesized based on the sequence of amino acids encoded on the mRNA.
Translation and Transcription
Translation and transcription are two sides of the same coin in prokaryotes. Even before a complete mRNA transcript is generated, translation of the mRNA begins. The simultaneous occurrence of these two processes is known as coupled transcription and translation. In contrast, these processes are spatially separated and occur sequentially in eukaryotic cells: transcription occurs in the nucleus, and translation takes place in the cytoplasm.
Once translated, polypeptides undergo various post-translational modifications to finalize their structure, determine their subcellular localization, and control their activity. Post-translational modifications, often known as PTMs, are chemical changes or additions made to the structure of a newly generated protein. These modifications play an essential role in cellular biology and other fields, such as peptide synthesis services.
Cloning Techniques for Protein Expression
Cloning is a critical factor in protein expression and purification that involves moving a piece of DNA or a gene of interest (GOI) from one organism to a genetic element that can replicate, such as an expression vector.
At a minimum, a standard expression vector consists of the following components:
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A bacterial origin of replication (ori)
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An antibiotic selection cassette for E. coli (e.g., conferring resistance to blasticidin S, Zeocin, ampicillin, or carbenicillin)
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An antibiotic selection cassette for a specific host (conferring resistance to Geneticin, Zeocin, Hygromycin B, puromycin, blasticidin S, or mycophenolic acid)
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A GOI expression cassette (comprising a promoter and a terminator or poly(A) signal)
The following elements may also be added:
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Epitope tags
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Internal ribosome entry sites (IRES)
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A multiple cloning site (MCS, or polylinker)
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Protease recognition sites
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Secretion signals
Most vectors already contain a promoter to facilitate expression in a particular host system.
Choosing an Expression System
A protein expression and purification protocol tailored to a particular application is essential to success. Vital criteria in selecting an expression system include protein solubility, functionality, purification speed, and yield. Furthermore, each system has its own benefits and drawbacks, which should be considered before settling on an expression platform. Usually, custom antibody and protein services offer six different ways to produce proteins: mammalian, insect, yeast, bacterial, algal, and cell-free systems.
Once a system is chosen, the method of gene delivery must be considered. Transfection and transduction are the two main ways genes are introduced to cells. Through transfection, nucleic acids are inserted into mammalian and insect cells. There are many different protocols and procedures, including lipid transfection and physical approaches like electroporation.
Virus-mediated Transfection
Viral vectors are frequently used in place of lipid-mediated transfection for cell types that are incompatible with lipid-based methods. In protein production services, virus-mediated transfection—sometimes referred to as transduction—provides a means to reach difficult-to-transfect cell types for protein overexpression or knockdown; it is the most widely used technique in clinical trials. Vectors such as adenoviruses, oncoviruses, lentiviruses, and baculoviruses have been used to deliver genes into mammalian cells in cell culture and living animals.
Purifying a protein of interest is one of the most critical steps in the workflow. The stages of protein purification usually consist of the following:
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Cell lysis (breaking up the cells)
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Attaching the protein to a matrix
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Washing
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Elution
Together, these processes enable biotechnology providers to produce several products like ELISA kits, and support services like gene synthesis for application in several scientific realms.
Cell Lysis
Cell lysis can be carried out using various techniques, such as non-enzymatic methods (including sonication or using a French press), hydrolytic enzymes like lysozyme, or detergent reagents such as FastBreak™ Cell Lysis Reagent. Due to the difficulty of purifying native proteins, affinity purification tags are frequently fused during recombinant protein production to trap or detect the protein.
Many factors, including the cell type used to create the recombinant protein, affect which purification method yields the highest quality protein (e.g., prokaryotic versus eukaryotic cells). Due to its versatility, rapid cell growth, and low culture cost, Escherichia coli is still the go-to organism for many scientists creating recombinant proteins. Relatively large amounts of proteins synthesized in E. coli can be purified, although these proteins, particularly eukaryotic proteins, might not exhibit appropriate protein function or folding. Alternatively, it may be possible to produce adequately folded, functional mammalian proteins with the necessary post-translational modifications using cultured mammalian cells.
Also, affinity purification tags may be attached to a target recombinant protein to streamline the purification process. Polypeptides, tiny proteins, or enzymes are frequently used as fusion tags and are attached to a recombinant protein's N- or C-terminus. Proteins that carry certain tags are more likely to be stable, soluble, and highly expressed due to those tags’ unique biochemical properties. Hence, using expression vectors with a fusion tag makes it easier to isolate recombinant proteins.
Affinity Tags For Protein
Affinity tags are a common way to identify a specific protein or immobilize it. There are many different kinds of affinity tags; these fusion tags are added to a recombinant protein’s N- or C-terminus and can be polypeptides, small proteins, or enzymes.
Glutathione-S-Transferase (GST)
The glutathione-S-transferase (GST) is an effective affinity tag because it binds firmly to glutathione-covered immobilized matrices. Eukaryotic organisms possess a family of proteins called glutathione-S-transferases that are found in the cytosol and have multiple functions. It is possible to purify GST-fusion proteins without interference from endogenous bacterial proteins because GST isoforms are not generally found in bacteria. Several eukaryotic proteins expressed in bacteria benefit from the addition of a 26 kDa GST affinity tag, which often increases their solubility.
Polyhistidine
By far, the most common type of tag is the polyhistidine tag (His-tag). The polyhistidine tag’s ability to purify proteins depends on the affinity of histidine residues for immobilized metals, such as nickel. It is hypothesized that this affinity contact occurs when nitrogen on the imidazole moiety of polyhistidine binds with an empty coordination site on the metal. By forming a complex with a chelate that is covalently bound to the support, the metal is anchored to the resin.
Compared to larger tags, the polyhistidine tag is less likely to provoke an immune response because of its diminutive size. Consequently, the tag usually does not need to be removed for downstream applications after purification. Finally, since the polyhistidine tag’s interaction with the metal is independent of the tag’s tertiary structure, it is possible to purify ordinarily insoluble proteins under denaturing conditions.
HaloTag® Protein Tag
Protein fusion tags are employed to facilitate purification and the production of sufficient quantities of soluble protein. The HaloTag® protein is a unique protein tag developed to improve the production and stability of recombinant proteins in E. coli. The HaloTag® protein tag is a monomeric protein tag with a molecular weight of 34 kDa derived from Rhodococcus rhodochrous dehalogenase.
The HaloTag® protein binds quickly and covalently to a unique synthetic linker to create a permanent bond. Labeling fusion proteins in cell lysates for expression screening and capturing fusion proteins on a purification resin is possible due to the synthetic linker’s ability to adhere to a wide range of entities, including fluorescent dyes and solid supports. HaloTag® technology is compatible with many protein production methods, including E. coli, mammalian cells, and cell-free systems.
Protein Purification Methods
Purification of GST-Tagged Proteins
An increasing number of researchers are looking for approaches applicable to the purification of recombinant proteins used in high-throughput screening. Magnetic resins make it possible to purify affinity-tagged proteins without the need for numerous centrifugation steps or transferring samples between tubes. Several characteristics make a suitable protein purification resin:
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It should exhibit minimal non-specific binding (i.e., it should not bind to non-target proteins).
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It should bind to the fusion protein efficiently.
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It should allow for easy recovery (elution) of the fusion protein.
These requirements are met by the MagneGST™ Protein Purification System, which makes it possible to purify proteins with a wide variety of molecular weights and expression levels. The magnetic properties of the binding particles enable purification using only a single tube, even when starting with crude lysate. In addition, high-throughput applications requiring automated liquid-handling platforms can take advantage of this technology. When designing protocols for automated workstations, we suggest using the manual protocol as a baseline.
Purification of Polyhistidine-Tagged Proteins
High-throughput methods for purifying proteins are becoming increasingly vital. Magnetic resins make it possible to purify affinity-tagged proteins without extensive centrifugation or the need to transfer samples from one tube to another. Several factors determine the suitability of a protein purification resin:
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It should not bind to non-target proteins.
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It should bind efficiently to the fusion protein.
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It should allow for simple recovery (elution) of the fusion protein.
These requirements can be satisfied by the MagneHis™ Protein Purification System, which makes it possible to purify proteins spanning a wide range of molecular weights and exhibiting various expression levels. Purification can be performed in a single tube from crude lysates due to the magnetic nature of the binding particles. In addition, high-throughput applications requiring automated liquid-handling systems can take advantage of this technology.
Purification of HaloTagged Protein
The conditions present in cultured mammalian cells are ideal for generating correctly folded and functional mammalian proteins with the necessary post-translational modifications. However, the modest levels of recombinant protein expression in cultured mammalian cells constitute a significant obstacle. Capturing these proteins efficiently and selectively from the crude cell lysate is essential for achieving optimal yield and purity.
Most methods of purifying proteins using affinity tags rely on equilibrium binding. In this process, the protein constantly moves back and forth between the bound (to the resin) and unbound (in the solution) states. The protein concentration and the tag’s binding affinity affect this equilibrium. As a result, binding effectiveness may be diminished at low expression levels, resulting in limited fusion protein recovery. You can genetically fuse the HaloTag® protein tag to any protein and then either transiently or stably express it in mammalian cells using the HaloTag® Mammalian Protein Purification Systems.
After cell lysis, the HaloTag® fusion protein is covalently captured on the HaloLink™ Resin, while non-specific proteins are removed by washing. The amino acid linker sequence that joins the HaloTag® protein tag to the protein of interest contains a specific proteolytic cleavage site (TEV recognition site) that releases the target protein. TEV protease fused to HaloTag® (HaloTEV Protease) can be utilized to cleave the HaloTag® fusion protein. The protease is then covalently trapped on the HaloLink™ Resin, eliminating the need for an additional step to remove the protease and resulting in a streamlined purification procedure.
Conclusion
There is much more to explore regarding the methods and strategies for protein expression and purification. However, we aimed to provide those just starting in the field with a resource based on popular approaches in recombinant protein production and peptide synthesis. By using these various methodologies, researchers can optimize their workflows to yield high-quality proteins for a wide range of applications.