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Considering the differences between bacterial and mammalian expression.
Recombinant proteins are widely used throughout biological and biomedical research. Every researcher using externally sourced proteins knows the importance of obtaining pure, high quality, uncontaminated product, with high consistency from batch to batch – such attributes are vital for obtaining reliable and reproducible results. Often overlooked when assessing protein quality from a manufacturer, though, is the expression system used.
Commercial protein production successfully uses many organisms as expression systems across the kingdoms: bacterial, plant, fungal, insect, or mammalian cell lines, including human.
If your research interests are in bacteria, then it makes sense to buy a protein produced in bacteria. But be cautious if you are conducting human research – many human-origin proteins are commercially produced in bacterial systems, which means they will not be folded or modified correctly, and so won’t function in the same way as their native-identical counterpart, nor be as stable. This can make research results less applicable to a real-world clinical setting.
“Mammalian expression systems have the cellular machinery to ensure the PTM (post-translational modifications) in commercial proteins are identical to those in that same protein expressed in the human body – but this machinery simply isn’t present in prokaryotic systems. Correct PTM also helps the protein to fold properly,” explains Dr Gráinne Dunlevy, Head of Protein Sciences at Abcam’s Cambridge Biomedical Campus in the UK and also Abcam’s research facility in Hangzhou, China. “If your assay needs a functional, human-origin protein which is native-identical, there’s a good chance the bacterially-expressed equivalent protein won’t work as well and you will have to add more protein.”
In the body, simplistically, nuclear DNA encoding a gene is transcribed into mRNA, translated into a polypeptide, and folds into the protein’s 3D structure, driven both by the inherent amino acids’ properties, as well as any PTM induced by the native environment. The resulting functional protein is secreted or transported to its ultimate destination.1
In the lab, a DNA construct encoding the target protein is transfected via a cloning vector into a biological expression system, and the resultant protein secreted into the culture medium. It is then captured using an affinity column, purified, and analyzed for purity and functionality.2,3 Although transcription and translation are similar across kingdoms, folding and PTM differ and depend largely on the native environment and cellular anatomy of the expression species.
In mammalian cells, folding occurs in the endoplasmic reticulum – a structure absent from bacterial cells – which contains enzymes and an oxidizing environment to facilitate proper folding, disulfide bond formation, and N-linked glycosylation.4 A protein destined for secretion would then pass into the Golgi apparatus (found only in eukaryotes) where O-linked glycosylation may occur (Figure 1).4
A protein that is not folded correctly and without its native PTM would not be functional or even stable, which is why larger, more complex proteins are best commercially expressed using mammalian systems.2
Despite the issues with correct folding and PTM discussed above, many protein manufacturers use bacteria to produce proteins of human origin. Certainly, there can be advantages in terms of protein yield and ease of production using prokaryotic versus mammalian systems.
“It’s all about what you need your protein for,” states Dr Dunlevy. “In a biochemical assay, such as a western blot, you just want to detect the presence and quantity of the protein, so a protein expressed in a non-mammalian system should be fine. However, for cell-based assays – such as those assessing viability, proliferation, cytotoxicity, or cell death – where functional bioactivity is important, it’s essential to use a protein identical to the human native protein. And that means expressing it in a mammalian system”.
Human-origin proteins made in prokaryotic systems are less stable than those generated in mammalian cell lines too – native glycosylation helps to hold the protein together correctly. Partially folded prokaryotic-produced versions of human-origin proteins are unstable, prone to degradation, and can be ‘sticky’ – ie they clump together to form aggregates. This can introduce variability into experiments and reduce their viable shelf-life.
However, even researchers early in discovery programs could benefit from using native-identical products from the very beginning. Switching products partway through development requires a lot of time and effort in validating results and repeating experiments. Prokaryotic-produced proteins tend to be less expensive gram for gram compared to mammalian versions – but they can be a more costly option than expected over the whole lifecycle of a research project when considering the cost of potential lack of functionality, and eventual necessary transitioning to a native-identical product. Also, the increased stability (and thus shelf-life) of mammalian proteins compared with their bacterial counterparts make them an attractive option at any stage of research – a little goes a long way.
Dr Dunlevy has worked for more than 16 years making proteins internally in pharmaceutical and biopharmaceutical companies. “Our scientists would ask me to make proteins they could actually use in their activity assays, with high enough quality, and folded correctly – because they just couldn’t find them to buy commercially,” she says. “We saw this time and again. There’s a real gap in the commercial protein market for high-quality proteins, but researchers without the resources of big pharma just have to make do with what they can buy. That experience drove us at Abcam to want to make available superior quality human proteins, fit for bioactivity assay purposes.”
And so, our premium bioactive grade proteins were born – a range of small proteins expressed exclusively in mammalian systems, to suit the needs of researchers who want functional, native-identical human proteins with optimal bioactivity, exerting the right biological effect.
Our premium bioactive grade proteins range currently comprises 68 secreted human cytokines, and we plan to expand the catalog with more types of human proteins in the future.
1. Ribosomes, Transcription, and Translation. Essential of Cell Biology. https://www.nature.com/scitable/ebooks/essentials-of-cell-biology-14749010/122996756/. Accessed July 29, 2020.
2. Dalton, A.C., Barton, W.A. Over-expression of secreted proteins from mammalian cell lines. Protein Sci. 23, 517–525 (2014). https://doi.org/10.1002/pro.2439
3. Gräslund, S., Nordlund, P., Weigelt, J. et al. Protein production and purification. Nat Methods. 5, 135–146 (2008). https://doi.org/10.1038/nmeth.f.202
4. Aebi, M. N-linked protein glycosylation in the ER. Biochim Biophys Acta - Mol Cell Res. 1833, 2430–2437 (2013). https://doi.org/10.1016/j.bbamcr.2013.04.001