Recombinant protein expression methods.
Darriene Lois Suvilla provides an insight into the methods of recombinant protein expression.
Proteins can be synthesized and regulated depending on the functional requirement in the cell. DNA usually stores the blueprints for proteins, and regulated transcriptional processes can decode them to messenger RNA. The message coded by messenger RNA can then be translated into a protein. Keep in mind that transcription refers to the information transfer from DNA to messenger RNA. Translation is when proteins are synthesized depending on a sequence specified in the messenger RNA.
Transcription and translation in eukaryotes and prokaryotes
In prokaryotes, the transcription and translation processes usually happen simultaneously. The translation of messenger RNA begins even before the messenger RNA transcript can be fully synthesized, so the simultaneous transcription and translation of genes are called coupled transcription and translation.
The process may be spatially separated and happen sequentially, with transcription in the nucleus for eukaryotes. It can also occur in the cytoplasm in what is called protein synthesis.
In most cases, proteomics research can involve investigating all aspects of a protein, such as structure, modifications, function, protein interactions, or localization. Therefore, many protein expression services may need to find means of manufacturing or producing functional proteins of their interest to investigate how proteins regulate biology.
Because of the complexity and size of proteins, chemical synthesis is not a suitable option for this task. Instead, it would help if you had living cells with their cellular structure to harness as factories to create and construct proteins depending on supplied genetic templates.
DNA is easy to construct synthetically, unlike proteins, which are created in vitro using established recombinant DNA methods. Hence, DNA templates of particular genes with or without affinity tag sequences or add-on reporters may be built as templates for protein expression. DNA templates that produce these proteins are known as recombinant DNA.
Traditional techniques for recombinant protein expression usually involve transfecting a cell with a DNA vector containing the template and culturing the cell to transcribe and translate the protein of interest. The cell is often lysed to get the expressed protein for future purification.
Both eukaryotic and prokaryotic in vivo protein expression systems are usually used. The choice of the system can depend on the protein type, the desired yield, and the need for functional activity.
These expression systems include insect, mammalian, bacterial, and yeast. Each system has advantages and disadvantages, so selecting the proper expression system for a specific application can be the key to a successful recombinant protein system.
Mammalian protein expression
Because of its physiologically important environment, you can use a mammalian expression system to produce proteins with native activity and structure. It can lead to a high level of post-translational processing and functional movement.
A mammalian expression system is usually a preferred system for expressing mammalian proteins. These systems can produce complex proteins, antibodies, and other proteins for use in a functional cell-based assay. However, these benefits come with extra-demanding cultural conditions.
You can use a mammalian expression system to make proteins transiently or even through stable cell lines, meaning the expression construct can be integrated into the host's genome. While you can use regular cell lines over multiple experiments, transient production may produce large amounts of protein in a week to two weeks.
This transient and high-yield mammalian expression system uses suspension cultures, and you can make gram-per-litre yields. Besides, these proteins can have more post-translational modifications and native folding.
Insect protein expression
Insect cells can be utilized for high-level protein expression with modifications similar to those of a mammalian system. Several techniques can be used to produce recombinant baculovirus to express proteins of interest in an insect cell.
These systems may be easily scaled up and even adapted to high-density suspension culture for large-scale protein expression that is perhaps more functionally similar to native mammalian protein. While yields may be up to 500 mg/L, recombinant baculovirus can sometimes be time-consuming. Also, cultural conditions can be challenging compared to the prokaryotic systems.
Bacterial protein expression
A bacterial protein expression system is popular because it is simple to culture bacteria. They also grow fast and even produce recombinant proteins in high yields. However, multi-domain eukaryotic proteins expressed in bacteria can usually be non-functional, as the cells are not designed to provide the needed post-translational modifications for molecular folding.
Also, most proteins can become insoluble as inclusion elements that are hard to recover without harsh denaturants and cumbersome protein-refolding processes.
Cell-free protein expression
Cell-free protein expression refers to the in vitro synthesis of proteins utilizing translation-compatible extracts of entire cells. Whole-cell extracts can contain the macromolecules and components required for transcription, translation, and post-translational modification. These components can include regulatory protein factors, RNA polymerase, transcription factors, and many more.
These extracts may synthesize the protein of interest in just a couple of hours. While this is not sustainable for large-scale production, it is worth noting that in vitro translation or cell-free protein expression systems can have several benefits over the usual in vivo systems. Cell-free expression can allow for fast synthesis of these recombinant proteins without problems with cell culture.
A cell-free system can enable protein labelling using amino acids and the expression of proteins that go through rapid proteolytic degradation in intracellular proteases. Further, with the cell-free technique, expressing several different proteins simultaneously, like protein mutations, can be easier if you represent them on a small scale from various recombinant DNA templates.
Chemical protein synthesis
Chemical protein synthesis can be used for applications needing proteins labelled with unnatural amino acids. These proteins can be labelled at particular sites or even considered toxic to some natural expression systems.
Remember that chemical synthesis can produce pure protein, which works well for small peptides and proteins. However, the yield is also low with chemical synthesis.
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About the Author
Darriene Lois Suvilla is a freelance journalist.