Recombinant Protein Expression in Mammalian Cells: Techniques and Applications

Proteins are essential biomolecules that perform a variety of functions in living organisms. Recombinant proteins, which are produced by cloning and expressing genes in host cells, have become an indispensable tool in research, biotechnology, and medicine.

Mammalian cells are often preferred hosts for the expression of a protein of interest due to their ability to perform proper protein folding, post-translational modifications, and secretion.

In this article, we will discuss the techniques and applications of recombinant protein expression in mammalian cells – from the industrial use to the production of recombinant antibodies.

• The Clinical Standard: Recombinant protein expression in mammalian cells—specifically Chinese Hamster Ovary cells—is the industry gold standard for therapeutic antibody development due to their ability to produce human-like post-translational modifications.
• Mitigating Translation Risk: Transitioning directly to CHO cells during early-phase discovery minimizes the batch-to-batch variability and sequence liabilities often encountered when scaling from alternative hosts (like HEK or E. coli) to clinical production.
• Addressing Bottlenecks: Modern expression platforms integrate high-throughput (HTP) transient transfection, enabling biotechs to validate AI-designed antibody libraries rapidly without sacrificing yield or quality (maintaining >95% purity and <1 EU/mg endotoxin levels).

What is recombinant protein expression?

The expression of recombinant proteins in mammalian cells involves the transfer of a foreign gene into a mammalian host cell, which then synthesizes and processes the encoded protein according to the recombinant DNA template. This process is a fundamental tool in molecular biology research, protein production, and drug development.

Why Choose Mammalian Cell Expression Systems for Therapeutic Antibodies?

Mammalian cell cultures offer decisive biochemical advantages over prokaryotic hosts, primarily due to their sophisticated intracellular machinery. Mammalian systems, particularly CHO cells, execute complex post-translational modifications such as human-compatible glycosylation, phosphorylation, and sulfation.

These modifications do not merely ‘affect’ the protein; they strictly dictate vital pharmacokinetic properties, structural stability, and immune effector functions such as ADCC (Antibody-Dependent Cellular Cytotoxicity) and CDC (Complement-Dependent Cytotoxicity), which are non-negotiable for clinical efficacy.

Steps in Recombinant Protein Expression in Mammalian Cells

In the production of recombinant proteins, various production steps have to be carried out with the greatest diligence – be it the generation of cDNA, the insertion of recombinant DNA with an expression vector, protein synthesis, fermentation, protein purification, characterization or chromatography.

Optimization and enhancement is necessary, as it can be time consuming to achieve a high protein yield of a target protein with a high throughput (e. g. by the use of chaperones, IPTG or SDS-PAGE).

Additionally, effects like unwanted overexpression, aggregation and the consequent build of inclusion bodies that would affect product quality have to be prevented, which is why extensive knowledge in proteomics is needed.

Also, great attention has to be paid to solubility, as the production of soluble proteins is highly complex. But how are proteins changed to recombinant proteins?

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Plasmid Design and Cloning

The first step in recombinant protein expression in mammalian cells is the design and cloning of the plasmid. This plasmid contains the gene of interest, a promoter, and other regulatory reagents that control the expression of the gene. The plasmid is then transfected into the mammalian host cell.

Transfection

Transfection is the process of introducing the plasmid into the host cell, either performed via stable or transient. Stable transfection involves the integration of the plasmid into the host cell’s genome, resulting in long-term expression of the recombinant protein.

Transient transfection, on the other hand, involves the expression of the recombinant protein for a shorter period of time.

After transfection, the single amino acids usually undergo translation and transcription by the means of mRNA (messenger RNA), in order for the ribosomes in the cell’s cytoplasm (intracellular liquid) to commence gene expression.

Selection

After transfection, the host cells are selected for those that have taken up the plasmid and are expressing the recombinant protein. This is achieved through the use of selection markers such as antibiotics or fluorescent proteins.

Optimization

The expression of recombinant proteins can be optimized through the use of various techniques, such as the optimization of the promoter, codon optimization, and the use of fusion tags.

For early-stage biotechs facing runway risks, validating in silico AI antibody designs in the wet lab is a critical bottleneck. Traditional optimization is too slow. By utilizing high-throughput, direct-to-CHO transient expression platforms, researchers can express and screen hundreds to thousands of variants in parallel. This rapid turnaround provides actionable, reliable developability data early in the pipeline, eliminating dead-end candidates before costly scale-up efforts begin.

Applications of Recombinant Protein Expression in Mammalian Cells

There are various fields of application of recombinant protein expression in mammalian cells. Here are some examples of where the technology is in use.

Biopharmaceutical Production

Recombinant proteins are used in the production of biopharmaceuticals, including monoclonal antibodies, recombinant antibodies (designed to target a specific antigen), cytokines, and growth factors.

Mammalian cell expression systems are frequently used for the production of biopharmaceuticals due to their ability to perform post-translational modifications and protein folding properly.

As pipelines shift toward complex, multi-specific therapeutics, standard expression systems frequently falter, plagued by heavy/light chain mispairing, low yields, and aggregation. Overcoming these internal bottlenecks requires acting as a seamless extension of the lab.

Specialized CHO transient expression platforms offer the absolute scientific flexibility required to optimize vector ratios and expression conditions for bispecifics, ensuring >95% purity and scalable consistency that dramatically reduces translation risk from discovery to clinical phases.

Research Applications

Recombinant proteins are essential tools in molecular biology research, allowing scientists to conduct assays for studying the function and interaction of proteins. Mammalian cell expression systems are regularly used for research applications due to their ability to produce properly folded and functional proteins.

Industrial Applications

Recombinant proteins are used in a variety of industrial applications, including the production of enzymes, food additives, and industrial enzymes. Mammalian cell expression systems are often preferred for industrial applications due to their ability to produce large quantities of high-quality protein.

Recombinant protein expression systems – an overview

Expression systems – whether prokaryotic or eukaryotic – are used to produce recombinant proteins for various applications in biotechnology, pharmaceuticals, and research. While mammalian cell lines are widely used for recombinant protein production due to their ability to produce complex, correctly folded proteins, other expression systems have also been developed.

One commonly used expression system to produce heterologous proteins is bacterial cells, such as E. coli (Escherichia coli). Bacterial systems are fast, easy to use, and cost-effective, making them ideal for producing large quantities of proteins for research and industrial applications.

However, bacterial cells may not be suitable for producing eukaryotic proteins or complex proteins that require post-translational modifications or protein folding. Also their metabolic load can be a limiting factor.^​1​

Another expression system is yeast cell culture, such as Saccharomyces cerevisiae or Pichia pastoris. Yeast cells can produce recombinant proteins with post-translational modifications, such as glycosylation, making them useful for producing recombinant proteins that require proper folding and modifications for their activity.

Baculovirus or insect cells, such as Sf9 or Sf21 cells, are also used as an expression system for recombinant proteins. They are able to produce large quantities of correctly folded proteins and can also perform post-translational modifications such as glycosylation. Insect cells are commonly used for producing viral vectors, vaccines, and other therapeutic proteins.

Plant-based systems, such as tobacco or lettuce, are also being explored as an alternative expression system for recombinant proteins. Plants can produce complex proteins, post-translational modifications, and have low risk of pathogen contamination, making them a potential alternative for large-scale production of therapeutic proteins.

Challenges when using mammalian cells

Mammalian cells are commonly used as expression systems for producing recombinant proteins due to their ability to carry out post-translational modifications and secrete correctly folded and functional proteins. However, the technology also brings along several challenges.

Mammalian cells are more difficult to work with compared to bacterial or yeast expression systems, as they require more complex culture conditions and are more susceptible to viral contamination. Additionally, the high cost of maintaining mammalian cell lines and achieving high yields of protein production are significant challenges.

Moreover, the potential for immunogenicity of recombinant proteins produced in mammalian cells must be carefully considered. Finally, the ethical issues surrounding the use of mammalian cells in research must also be taken into account.

While mammalian cell culture presents inherent complexities, the strategic use of Chinese Hamster Ovary cells effectively neutralizes these risks. Because CHO cells have been cultured in vitro for decades, they offer a highly characterized, robust environment that requires no in vivo animal modeling.

The evitria Advantage in CHO Expression

At evitria, a premier provider of recombinant antibody production service and high-throughput antibody production service, we do not just use CHO cells we have spent 15 years exclusively engineering and perfecting transient expression within the CHO platform. Having successfully completed over 140,000 transfections and expressed more than 25,000 distinct antibodies, our platform is purpose-built to deliver peerless reliability. By providing endotoxin levels strictly <1 EU/mg and purity exceeding 95%, we empower our partners—from agile startups to top-tier global pharma—to accelerate their development pipelines with absolute scientific confidence.

Conclusion: What’s to know on recombinant protein expression in mammalian cells

Recombinant protein expression in mammalian cells is a powerful tool in research, biotechnology, and medicine. Mammalian cells often are preferred hosts for the expression of recombinant proteins due to their ability to perform post-translational modifications, protein folding, and secretion.

Techniques such as plasmid design and cloning, transfection, selection, and optimization are used to achieve efficient and high-yield recombinant protein expression.

The applications of recombinant protein expression in mammalian cells are numerous and include biopharmaceutical production, research applications, and industrial applications.

In order to overcome challenges related to the use of mammalian cells in recombinant protein production, we at evitria rely on the use of CHO cells. This way, we are able to provide partners with high quality recombinant proteins for various needs – from small scale to industrial applications.

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