In the dynamic realm of biotechnology, mammalian cell protein expression has emerged as a pivotal technology. Renowned for generating proteins highly similar to human forms, it's essential in research, therapeutics, diagnostics, and industry. From precise antibodies to intricate glycosylated proteins, it offers unique opportunities. This article explores the latest techniques and applications, revealing how optimizing it can lead to novel breakthroughs.

Why Mammalian Cell Expression?

Mammalian cell expression systems stand out because they provide the ability to produce complex proteins with human-like post-translational modifications. These modifications include glycosylation, phosphorylation, and proper protein folding, essential for many therapeutic proteins’ functionality, efficacy, and safety. Systems like E. coli or yeast often struggle to replicate these intricate modifications, making mammalian cells the preferred choice when accuracy and bioactivity are critical.

What are Applications of Mammalian Expression?

• Biopharmaceuticals: Mammalian expression systems are widely used in producing therapeutic proteins, including monoclonal antibodies, cytokines, and hormone replacements.

• Research and Diagnostics: The ability to express human-like proteins allows researchers to study disease mechanisms, conduct drug screening, and develop diagnostic tools.

• Industrial Biotechnology: In food and cosmetics, recombinant proteins produced in mammalian cells contribute to creating products that are safer, more effective, and of higher quality.

Essential Process of Protein Expression in Mammalian Cells

To achieve high-quality protein production in mammalian cells, the process involves a series of meticulous steps, from plasmid design to protein detection. Here’s a detailed look at each stage:

1. Plasmid Construction: Laying the Foundation

• Gene Cloning: Begin by isolating or synthesizing the gene of interest. PCR is commonly used to amplify this gene, with restriction sites added to facilitate vector insertion.

• Vector Selection: Choose a mammalian expression vector equipped with a strong promoter (e.g., CMV) and essential elements like polyadenylation signals and antibiotic resistance markers, which support stable protein expression and enable cell selection.

• Insertion and Amplification: After inserting the gene into the vector, transform it into E. coli for amplification, followed by plasmid purification before transfection into mammalian cells .

2. Cell Culture: Growing the Ideal Host

• Cell Line Selection: HEK293 and CHO cells are popular for their transfection efficiency and scalability. HEK293 cells are favored for research applications, while CHO cells are often chosen for large-scale therapeutic production.

• Optimizing Culture Conditions: Maintaining cells in a controlled environment (37°C, 5% CO₂) with nutrient-rich media such as DMEM or RPMI-1640 is essential. Fetal bovine serum (10-20%) is often added to support cell growth and protein expression.

3. Transfection: Delivering the Gene

• Transfection Methods: Several methods are available, each with its advantages:

• Lipid-Based Transfection: Lipid reagents like Lipofectamine encapsulate DNA, allowing it to enter cells via endocytosis. This is a high-efficiency, low-toxicity option.

• Electroporation: This uses an electric pulse to open cell membranes temporarily, allowing DNA to enter. It’s suitable for various cell types, though it requires careful optimization to prevent cell damage.

The chosen method will depend on the specific cell type, required expression level, and experimental goals.

4. Selection and Expansion of Transfected Cells

• Antibiotic Selection: If the vector contains a resistance gene, add the corresponding antibiotic (e.g., G418 for neomycin resistance) to the culture to isolate transfected cells.

• Expansion: Once colonies of transfected cells are established expand the cultures to increase cell numbers, ensuring adequate protein production for downstream applications.

5. Protein Expression and Detection: Confirming Success

• Expression Monitoring: After transfection, monitor the cells for protein expression. The expression level can be affected by factors like promoter strength and the plasmid’s copy number.

Detection Methods:

• Western Blotting: A classic and reliable method to verify protein size and quantity.
• ELISA: Highly sensitive and suitable for quantifying proteins in the supernatant or cell lysate.
• Immunofluorescence: Ideal for visualizing protein localization within cells, offering insights into how and where the protein is expressed.

Mammalian Expression | Synbio Technologies

In protein expression, researchers face issues like low yields, degradation, and cytotoxicity. These drive our optimization. Via codon optimization, fusion tags, medium tweaks, and culture condition adjustments, we break bottlenecks, advancing mammalian cell expression.

Synbio Technologies' Mammalian Expression Platform offers diverse services from high-throughput to large-scale recombinant antibody and recombinant protein production (mg - g). It provides highly pure (≥98%) recombinant proteins with＜0.001 EU/μg endotoxin. The mammalian cell systems ensure correct folding and post-translational mods, making products highly similar to natural ones in structure, properties and functions.