Learn Everything About Recombinant Protein Expression Methods

By  //  June 9, 2021

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Proteins can be synthesized and regulated depending on the functional requirement in the cell. DNA usually stores the blueprints for proteins and they can be decoded by regulated transcriptional processes to give 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. Translation is when proteins are synthesized depending on a sequence that is specified in the messenger RNA. This page discusses everything about recombinant protein expression methods.

Transcription and translation in eukaryotes and prokaryotes

In prokaryotes, the transcription and translation process usually happens simultaneously. The translation of messenger RNA begins even prior to messenger RNA transcript can be fully synthesized. So the simultaneous transcription and translation of genes is called coupled transcription and translation.

When it comes to eukaryotes, the process may be spatially separated and happen sequentially with transcription that occurs in the nucleus. It can also happen in the cytoplasm in what is called protein synthesis.

In most cases, proteomics research can involve you 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, you need 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 constructing 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 constructed as templates when it comes to 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 that has the template and culturing the cell to enable it 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 needs for functional activity.

These expression systems include insect, mammalian, bacterial, yeast, and many more. Each of these systems have their advantages and disadvantages, so selecting the proper expression system for a specific application can be the key for a successful recombinant protein system. 

Recombinant protein expression systems 

As explained earlier, there are many recombinant protein expression systems. Here are some of these systems:

Mammalian protein expression

You can use a mammalian expression system to produce proteins that feature native activity and structure because of its physiologically important environment. This can lead to a high level of post-translational processing as well as functional activity.  

A mammalian expression system is usually a preferred system to express mammalian proteins and you can use them for the production of 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 genome of the host. While you can use stable 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-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 have modifications similar to a mammalian system. You can use several systems to produce recombinant baculovirus that can be used to express proteins of interest in an insect cell.

These systems may be easily scaled up and even adapted to a high-density suspension culture when it comes to 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’s easy to culture bacteria. They also grow fast and even produce recombinant proteins of high yields. But multi-domain eukaryotic proteins that are 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 using harsh denaturants and cumbersome protein-refolding processes that follows.

Cell-free protein expression

Cell-free protein expression refers to the in vitro synthesis of proteins utilizing translation-compatible extracts of entire cells. Ideally, whole cell extracts can have the macromolecules as well as 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 when it comes to large scale production, it’s worth noting that in vitro translation or cell-free protein expression systems, they 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 any need of having problems with cell culture.

A cell-free system can enable protein labeling using amino acids and 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 at the same time like protein mutations if you express them on a small scale from various recombinant DNA templates.  

Chemical protein synthesis

Chemical protein synthesis can be utilized for applications needing proteins labeled with some unnatural amino acids, meaning these are proteins that are labeled at particular sites or even proteins that are considered to be toxic to some biological 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.

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