Selecting an Optimal Protein Expression System

Proteos Mammalian Cell Culture for Recombinant Protein Expression

Factors to consider when selecting the optimal expression system for your protein of interest:

Structural Complexity

The structural complexity of a protein can dictate which expression system is best suited for its recombinant production. A bacterial expression system is the clear choice for production of a recombinant protein of prokaryotic origin. Bacterial expression is also ideal for a eukaryotic protein with a simple tertiary structure that contains a single structural domain. Eukaryotic proteins with complex tertiary and/or quaternary structures are best suited for an insect or mammalian expression system that is equipped with the machinery for proper protein folding and post-translational modifications.

Post-Translational Modifications

Post-translational modifications (PTMs) can be critical for a protein’s folding, function, and localization. This includes, but is not limited to phosphorylation, glycosylation, prenylation, disulfide bonds, and protease cleavage. Bacteria lack many of the PTMs present in eukaryotic cells. As a result, heterologous expression of a eukaryotic protein in bacteria may yield a misfolded and/or functionally inactive product. Alternatively, recombinant production in insect cells provides a majority of the eukaryotic PTMs. However, certain processes, such as glycosylation, may be simplified in comparison to that of mammalian cells. Of the three expression systems, mammalian cells have the most sophisticated PTM pathways. When producing a eukaryotic protein that requires PTMs for its folding and function, a mammalian expression system is the optimal choice.

Mammalian cell expression culture in shake flasks, HEK-293, CHOSolubility

Solubility in bacteria can be a challenge when attempting to express a eukaryotic protein with a complex tertiary structure. Disulfide bond formation is difficult to achieve in bacterial cells, since the cytoplasm is a reducing environment. Furthermore, bacteria do not have the same chaperone network as eukaryotes to assist with correct folding. Consequently, eukaryotic proteins recombinantly produced in bacteria may be biologically inactive and localized to insoluble inclusion bodies. In some cases, it is possible to recover the protein of interest from inclusion bodies through solubilization and refolding steps. Oftentimes, it is more practical to optimize bacterial expression conditions, or switch to a eukaryotic expression system that is better equipped to produce the protein in its native state. 

Cellular localization

A bacterial, insect, or mammalian cell system can be a viable option for recombinant expression of an intracellular protein, when considering the criteria described above. Extracellular proteins, on the other hand, present additional challenges. Secretion to the oxidative extracellular space can be essential for the biological activity of proteins that require disulfide bonds for proper folding. Using a secretion signal, a prokaryotic protein of interest can be successfully targeted to the bacterial periplasm or extracellular space. However, secretion is rarely achieved for eukaryotic proteins that are heterologously expressed in bacteria, since they easily overwhelm the simple trafficking machinery. The intricate PTM and secretory pathways of eukaryotes make insect and mammalian expression systems the optimal choice for recombinant proteins targeted to the extracellular space.


Recombinant protein production in bacteria includes fast growth rates and simple culture conditions. Expression in insect and mammalian cell systems involves longer growth times and more expensive culture reagents. However, this does not automatically mean that bacterial expression is a better investment than the other two systems. Bacterial cells are a good starting point for a protein expression project, but the protein of interest may be consistently insoluble in this system. Switching to an insect or mammalian expression system may be a more efficient and cost-effective investment than continuing to troubleshoot in bacteria. Most importantly, choosing an expression system that is well suited for the downstream purpose of the recombinant protein ensures the best investment of valuable time and money. With optimized and validated protocols, each of these expression systems can be used to produce high yields of recombinant protein that is fit-for-purpose.

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Tags: E coli, expression, insect, mammalian, recombinant protein production

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