Mammalian cell expression systems are the gold standard for producing recombinant proteins that require complex post-translational modifications and biologically accurate folding. They play a crucial role in drug development, where biologics and monoclonal antibodies are manufactured, and heavily used in structural biology for producing proteins required in X-ray crystallography, cryo-electron microscopy, and NMR studies.
Despite their widespread use, mammalian expression systems are more complex and resource-intensive than bacterial or insect cell systems. Although they require specialized culture conditions, their ability to generate fully functional, biologically relevant proteins makes them the preferred choice for applications where protein authenticity is critical.
How Mammalian Cell Expression System Works
Mammalian cell expression systems rely on transient or stable transfection to introduce the gene of interest into the host cells. The choice between these two approaches depends on the experimental requirements and production scale.
Mammalian cells can be cultured as either adherent or suspension cells. Adherent cells grow attached to a solid surface and are commonly used for small-scale or early-stage experiments due to their ease of handling and compatibility with standard lab equipment. In contrast, suspension cells grow freely in liquid media and are better suited for large-scale protein production, as they can be cultured in bioreactors and scaled up more efficiently. Most industrial protein expression workflows now favor suspension-adapted cell lines due to their higher yield and process scalability.
1. Gene Delivery and Transfection
The first step involves inserting the target gene into an expression vector, typically under the control of a strong mammalian promoter, before being introduced into mammalian cells via chemical (lipid-based transfection), electroporation, or viral transduction (e.g., lentivirus or adenovirus).
Transient Transfection: DNA is not integrated into the genome, leading to rapid but temporary protein expression (lasting days), suitable for small-scale research applications.
Stable Transfection: The DNA integrates into the genome, allowing for long-term protein production.
2. Stable Isotope Labeling
To produce isotope-labeled proteins, mammalian cells are grown in media supplemented with stable isotope-labeled nutrients, such as ¹³C-labeled glucose, ¹⁵N-labeled amino acids, or deuterated components. These labeled precursors are incorporated into proteins during biosynthesis, enabling downstream structural and functional studies using NMR or mass spectrometry. In addition to traditional defined media, rich media formulations containing stable isotope-labeled amino acids are now available. Silantes has developed and recently commercialized such rich media, offering researchers enhanced cell growth and protein yields without compromising labeling efficiency for demanding isotope-labeling workflows.
3. Protein Folding and Post-Translational Modifications
Mammalian cells possess the necessary machinery for proper protein folding, glycosylation, phosphorylation, and disulfide bond formation, ensuring functional protein production. These modifications are essential for therapeutic proteins, as they influence stability, activity, and immunogenicity.
4. Harvesting and Purification
Once sufficient protein expression is achieved, the cells are lysed, and proteins are extracted and purified using techniques such as affinity chromatography, ion-exchange chromatography, and size-exclusion chromatography. Analytical techniques, including mass spectrometry, confirm isotope incorporation and protein purity.
Source – https://link.springer.com/article/10.1007/s00216-010-3821-6/figures/1
Advantages & Disadvantages of Mammalian Cell Expression Systems
Mammalian expression systems provide the highest level of protein authenticity but come with significant costs and technical challenges. The table below summarizes the key advantages and disadvantages of this system:
| Silantes Stable Isotope-Labeled Medium | Amino Acid Mixture Medium | |
| Convenience | Ready-to-use, pre-formulated medium for minimal preparation time and reduced variability | Requires manual preparation and precise supplementation of multiple isotope-labeled amino acids that is time-consuming and error-prone |
| Isotopic Uniformity | Delivers comprehensive, uniform labeling across a wide range of proteins | Limited to selected amino acids, leading to incomplete or uneven labeling. |
| Data Quality | Produces consistently high-quality NMR and MS data due to uniform labeling and reduced metabolic scrambling. | Inconsistent labeling can complicate spectra interpretation and reduce data reliability. |
| Economic Efficiency | Optimized for cost-effectiveness at scale by using metabolically incorporated isotopes rather than expensive free amino acids. | High cost due to reliance on multiple, individually labeled amino acids, especially for complex or large-scale studies. |
| Application Versatility | Ideal for mammalian systems and compatible with CHO and HEK cells; supports studies of complex, post-translationally modified proteins. | Less suited for full protein labeling in mammalian systems; often used for simplified or partial labeling strategies. |
| Scalability | Designed for reproducible scale-up in industrial and research applications | Scalability limited by the manual nature of preparation and higher costs of inputs. |
In summary, mammalian cell systems are indispensable for producing high-fidelity proteins with human-like modifications, making them the leading choice for therapeutic protein production. However, their high cost, technical complexity, and lower yields must be carefully considered when selecting an expression system.
Optimizing Your Protein Expression with Mammalian Cell Systems
At Silantes, we offer cutting-edge mammalian expression systems designed for high-yield protein production in mammalian cells. Our solutions are ideal for producing biologics, antibodies, and complex recombinant proteins. Additionally, our bio-based stable isotope-labeled solutions provide enhanced capabilities for your research. Not sure which system is right for your research? Contact us today, and our experts will help you find the best solution for your needs.