The mammalian protein expression system is widely used for producing recombinant proteins that require proper folding, post-translational modifications (PTMs), and biological activity, similar to native proteins in human cells. Mammalian systems are particularly useful for expressing complex proteins like antibodies, membrane proteins, and proteins that require glycosylation, phosphorylation, or other PTMs that bacterial or yeast systems cannot perform accurately.
Here’s an overview of the key steps and considerations in using mammalian cells for protein expression:
Selection of Expression System
The most commonly used mammalian cell lines for recombinant protein expression are:
HEK293 (Human Embryonic Kidney 293 cells): Commonly used due to their high transfection efficiency and ease of handling.
CHO (Chinese Hamster Ovary cells): The most widely used cell line for industrial protein production (e.g., therapeutic antibodies), known for its ability to produce large amounts of properly glycosylated proteins.
NS0 or SP2/0: Murine myeloma cell lines, also used for therapeutic antibody production.
Cloning the Gene of Interest
Gene optimization: Optimize the gene of interest for mammalian codon usage to improve expression efficiency.
Expression vector: Insert the gene of interest into a mammalian expression vector that contains:
A strong promoter (e.g., CMV, EF1α, SV40) to drive high levels of transcription.
Enhancers to increase transcription efficiency.
Polyadenylation signals for mRNA stability.
Selectable markers (e.g., antibiotic resistance genes like puromycin or neomycin) for selecting stable transfectants.
Optional fusion tags (e.g., His-tag, FLAG-tag, Fc-tag) for easier protein purification.
Signal peptides: If the protein is secreted, include a signal sequence to direct the protein to the secretory pathway.
Transfection into Mammalian Cells
Transfection is the process of introducing the recombinant plasmid into mammalian cells. There are two types of transfection strategies:
Transient transfection: Used when only short-term expression is needed, such as for small-scale protein production or screening experiments. Transfected cells express the recombinant protein for a few days before the plasmid is lost.
Common methods: Lipid-based transfection (e.g., Lipofectamine), electroporation, or calcium phosphate transfection.
Advantages: Fast and suitable for small to medium-scale protein production (1–2 weeks from transfection to protein harvesting).
Disadvantages: Protein yields are usually lower, and the process is not ideal for large-scale production.
Stable transfection: Used for long-term protein production. The gene of interest is integrated into the host genome, enabling continuous protein expression over multiple cell generations.
Selection: After transfection, cells are subjected to selection with antibiotics (e.g., puromycin, neomycin, hygromycin) to isolate stable clones.
Advantages: Suitable for large-scale and long-term production, as stable cell lines can be maintained for months or years.
Disadvantages: Time-consuming and requires several weeks to establish stable cell lines.
Protein Expression and Optimization
Serum-free vs. serum-containing media: Mammalian cells are often cultured in media containing fetal bovine serum (FBS) to support growth. However, for protein production, serum-free media is preferred to avoid contamination with serum proteins.
Optimizing expression conditions: Factors such as temperature, CO₂ levels, media composition, and duration of expression need to be optimized. Lowering the temperature (e.g., from 37°C to 30°C) after transfection can improve protein folding and yield.
Secreted vs. intracellular proteins: If the protein is secreted, collect the culture medium. For intracellular proteins, the cells are harvested, lysed, and the protein is purified from the lysate.
Protein Purification
The purification method depends on the nature of the protein and whether it has a purification tag.
Affinity chromatography:
For proteins with tags like His-tag or FLAG-tag, affinity chromatography using nickel or cobalt resins (for His-tag) or anti-FLAG resin can be used to purify the protein.
For Fc-fusion proteins, Protein A or Protein G affinity chromatography is used to purify the protein based on the Fc region.
Ion-exchange chromatography:
Proteins can be purified based on their charge at a given pH using cation or anion-exchange chromatography.
Size-exclusion chromatography (SEC):
This method is used to separate proteins based on size and can help remove aggregates or multimers.
Purification from the culture supernatant (for secreted proteins):
If the protein is secreted, it can be purified directly from the culture medium. Serum-free medium is preferred for cleaner purification without interference from serum proteins.
Verification and Quality Control
Once the protein is purified, several methods are used to confirm its quality, quantity, and activity:
SDS-PAGE and Western blotting: Used to assess the purity and molecular weight of the protein.
Mass spectrometry: To confirm the identity of the protein and check for correct modifications (e.g., glycosylation).
Functional assays: Enzyme assays, binding assays, or other bioassays to confirm the biological activity of the recombinant protein.
Glycosylation analysis: If the protein is glycosylated, techniques like mass spectrometry or lectin-binding assays can be used to analyze glycosylation patterns.
Scale-up of Protein Production
For large-scale protein production, mammalian cell cultures can be scaled up using:
T-flasks: Suitable for small-scale protein production.
Roller bottles: Intermediate scale.
Stirred-tank bioreactors: Large-scale production for industrial purposes, enabling precise control of environmental parameters (e.g., pH, oxygen levels, nutrient supply).
Wave bioreactors: Another scalable system, where cells grow in a rocking bag setup, providing gentle mixing and aeration.
Glycosylation and Post-Translational Modifications
One of the primary advantages of mammalian systems over bacterial or yeast expression systems is their ability to perform human-like post-translational modifications, including:
N-linked and O-linked glycosylation: Critical for the activity, stability, and solubility of many therapeutic proteins, such as antibodies and enzymes.
Phosphorylation, acetylation, and methylation: Important for regulatory proteins or signaling molecules.
Disulfide bond formation: Essential for the structural stability of many secreted and membrane proteins.
Advantages of Mammalian Expression Systems
Post-translational modifications: Mammalian cells are the best system for producing proteins with proper glycosylation, phosphorylation, disulfide bond formation, and other modifications.
Folding: Proteins expressed in mammalian cells are more likely to fold correctly compared to bacterial or yeast systems, reducing the need for refolding procedures.
Functional relevance: Proteins expressed in mammalian systems are more likely to resemble their native forms, making them ideal for therapeutic or diagnostic use.
Disadvantages of Mammalian Expression Systems
Cost: Mammalian cell culture is significantly more expensive than bacterial or yeast culture due to the need for specialized media, equipment, and longer culture times.
Slower growth: Mammalian cells grow more slowly than bacterial or yeast systems, leading to longer production times.
Lower yields: Protein yields in mammalian systems are generally lower than in bacterial systems, especially in transient transfection systems.
Applications of Mammalian Expression Systems
Therapeutic proteins: Production of monoclonal antibodies, hormones, cytokines, and other therapeutic proteins that require correct folding and post-translational modifications.
Vaccine production: Mammalian cells are used to produce viral proteins or whole viruses for vaccines.
Structural biology: Producing proteins for crystallization and structure determination.
Functional studies: Expression of receptors, ion channels, and other membrane proteins for drug discovery.
Summary of Workflow:
- Clone gene of interest into a mammalian expression vector.
- Transfect mammalian cells (HEK293, CHO, etc.) using transient or stable transfection.
- Culture cells under optimal conditions and harvest the protein from the culture medium or cell lysate.
- Purify the protein using affinity, ion-exchange, or size-exclusion chromatography.
- Characterize the protein for purity, structure, and function.
- Scale up the production if large quantities are needed.
Mammalian systems are the go-to choice for producing complex proteins that need proper folding and post-translational modifications, making them indispensable for the development of biopharmaceuticals and other advanced protein-based research.