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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 that is 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. The translation is when proteins are synthesized depending on a sequence that is specified in the messenger RNA.

Transcription and translation in eukaryotes and prokaryotes

In prokaryotes, the transcription and translation process usually happens 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 is called coupled transcription and translation.

The process may be spatially separated and happen sequentially with transcription in the nucleus when it comes to 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 like structure, modifications, function, protein interactions or localization. Therefore, to investigate the way proteins regulate biology, many protein expression services may need to find means of manufacturing or producing functional proteins of their interest.

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 or creating in vitro utilizing 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 proteins.

Traditional techniques for recombinant protein expression usually involve transfecting a cell with a DNA vector with the template and culturing the cell to transcribe and translate the protein of interest. In most cases, the cell is lysed to get the expression protein for future purification.

Both eukaryotic and prokaryotic in vivo protein expression systems are usually used. The choosing 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, yeast, and many more. 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 that feature native activity and structure. This can lead to a high level of post-translational processing as well as functional movement.

A mammalian expression system is usually a preferred system to express mammalian proteins. You can use them to produce complex proteins, antibodies, and proteins for use in a functional cell-based assay. But 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 use suspension cultures and you can make gram-per-liter 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 that has modifications similar to a mammalian system. You can use several techniques 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 quite similar to native mammalian protein. While yields may be up to 500 mg/L, it means recombinant baculovirus can sometimes be time-consuming. Also, culture conditions can be challenging compared to the prokaryotic systems.

Bacterial protein expression

A bacterial protein expression system is popular simply because it is simple to culture bacteria. They also grow fast and even produce recombinant proteins of high yields. But multi-domain eukaryotic proteins expressed in bacteria can usually be non--functional as the cells are not designed to give the needed post-translational modification or 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 have the macromolecules and components required for transcription, translation, and post-translational modification. Remember that 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. This is because cell-free expression can allow for fast synthesis of these recombinant proteins without having 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, it can be easier to express several different proteins simultaneously, like protein mutations, 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 some unnatural amino acids. These are proteins labelled at particular sites or even proteins considered toxic to some natural expression systems.

Remember that chemical synthesis can produce pure protein, though it works well for small peptides and proteins. Yield is also usually pretty low with chemical synthesis.

Page Reference

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  • SUVILLA, D. (2021) Recombinant protein expression methods [WWW] Available from: [Accessed

About the Author

Darriene Lois Suvilla is a freelance journalist.