Mammalian Cell Expression System

Mammalian cell transient and stable expression systems

The main advantages of mammalian cell expression are that the signals for synthesis, processing and secretion of eukaryotic proteins are properly and efficiently recognized by the mammalian cells. Therefore, the recombinant proteins are folded and oligomerized very similar to their natural counterparts.

A recombinant protein produced by mammalian cells has a higher activity than the one produced by other systems (i.e. prokaryotic expression systems and eukaryotic expression systems such as yeast and insect cells), and is close to a naturally produced one. This makes the mammalian expression systems widely used in developing and producing protein medicines, and in particular the therapeutic monoclonal antibodies.

The protein expression using mammalian cells includes two types: transient expression and stable expression. The biggest difference between the two expression methods is the time required. For transient expression, protein expressing cells can be harvested four days after transfection. Transient expression is generally used for short-term expression of gene products and small-scale protein synthesis. The technological breakthroughs on cell culture and transient transfection have enabled people to generate recombinant proteins in large-scale. Compared to transient expression, stable expression is suitable for functional studies, long-term pharmacological research and genetic regulation mechanism research. The stable expression is bottlenecked by the generation of stable cell lines, which are labor and time consuming.

Transfection methods for mammalian cells

Cell transfection refers to a technique of introducing exogenous genes into cells. Currently, commonly used methods of cell transfection mainly fall into three categories: physical methods (electroporation, gene gun, microinjection), chemical methods (liposome transfection, calcium phosphate co-precipitation, cationic polymer-mediated transfection), and biological methods (virus-mediated transfection, protoplast transfection). The ideal cell transfection method should have benefits such as high transfection efficiency and low cytotoxicity.

The table below shows the principles, advantages, disadvantages, and applicability of some commonly used transfection methods:

Cell Transfection Methods Transfection Principle Main Characteristics
Cationic Liposome Transfection Method Cationic liposomes and negatively-charged DNAs form DNA-liposome complexes through electrostatic interactions, which then enter cells by endocytosis · Easy operation
· Good for various uncovered DNA and RNA fragments
· Suitable for transfecting various cells
· A minimal DNA concentration is required
· Somewhat toxic to cells
Cationic Polymer Transfection Positively-charged cationic polymers form complexes with the negatively-charged phosphate groups of nucleic Cationic polymers are similar to the liposomes, but with low toxicity. It is a new generation transfection reagent
Calcium Phosphate Transfection Method Calcium phosphate can promote the binding of exogenous DNA to cells. The calcium phosphate-DNA complex enters the cell through endocytosis · Simple operation
· Requirement of a high DNA concentration
· Limited applicability (e.g. unsuitable for primary cells)
Electroporation Method An electrical pulse is used to create temporary pores in cell membranes through which DNAs can pass. After electroporation, the cell membrane recovers, and expression of the transfected nucleic acid can occur · Wide applicability, suitable for plasmids and gene fragments of sizes up to tens of kilobases
· Experimental conditions need to be optimized for different cells
· High cell death rate
Microinjection Method DNA is directly injected into cells using a microinjection system and technique · High integration rate, suitable for engineering and establishing transgenic animal models
· Special and expensive instruments are required
· The integration site and copy number of foreign genes cannot be controlled, which can lead to fragment deletion and mutation
Viral Transfection Method DNAs are transferred into cells through the interaction between virus membrane proteins and cell surface receptors. The transcription and replication of foreign DNAs are initiated by host cell enzymes; foreign DNAs randomly integrate into the cell genome · High transfection efficiency, suitable for transfecting difficult-to-transfect cells
· By using virus mediated transfection, the integration of foreign genes is relatively stable
· Retroviruses selectively infect dividing cells
· Foreign gene lengths are normally limited to a size less than 8kb
Construction of stable cell lines in mammalian cells

A mammalian stable cell line is constructed by integrating foreign genes into the cell's own genome, allowing foreign genes to become a part of the cell genome. A stable cell line is built upon transiently transfected cells, which are screened according to their resistance markers on plasmids. Afterwards, cells were passaged by the selective drugs, and cells that have not been stably integrated by foreign genes are eliminated. Stably transfected monoclonal cell line was further obtained by flow cytometry or limited dilution. Stable cell lines play an important role in producing recombinant proteins/antibodies, genome editing, and functional assays.

Demands and solutions for mammalian stable cell lines
Requirement Solution
Integrating foreign genes into a cell’s chromosomes Insert genes through genome editing and other methods
Reduce genotype differences between individual cells of the same type of cells. Because these differences interfere with the experimental results Screening for stable monoclonal cell lines
Foreign genes are not successively integrated into cells of an recipient animal, resulting in loss of foreign genes or their segments Cells with integrated foreign genes need to be injected into the body of the recipient animal
RNA interference exerts little inhibitory effects on the target gene Screening stable cell lines with better gene silencing effects
Obtaining efficient expression of a foreign gene or its segment Screening stable cell lines with better gene expressing effects
Increasing expression of a target gene or copy number of an interfering gene Screening stable cells with appropriate copy numbers
AlpHelix provides protein expression services using transiently transfected mammalian cells

Scientists of AlpHelix have rich experience in molecular biology and engineering of mammalian cells. Our proprietary High Density (HD) mammalian cell transient expression system, as well as expression protocol and cell culture media, ensuring the production of recombinant proteins and monoclonal antibodies at gram levels. We can provide flexible services to meet different needs, including removal of affinity tags and endotoxin, as well as glycosylation analysis, etc.

1-2weeks 1-2weeks 2-3weeks 3-4weeks
Gene synthesis and codon optimization (optional) Vector construction Pilot protein expression and purification Large-scale expression and purification (optional)
Customer provides protein sequence information or plasmid · Cloning into optimized high-efficiency expression vector of AlpHelix
· Plasmid sequencing
· Plasmid preparation

Deliverables
· Sequencing report (if requested by the customer) 		· Transient transfection of HEK293/CHO cells
· Protein purification in small-scale
· QC analysis (SDS-PAGE, UV, etc.)

Deliverables
· 0.1-0.5mg purified protein (if protein is stable and soluble)
· Certificate of analysis(COA) 		· 100mg or more protein
· QC analysis

Deliverables
· Purified protein
· Certificate of analysis(COA)
AlpHelix provides protein expression services using mammalian stable cells

Stable cell lines have wide applications, such as producing recombinant proteins and antibodies, genome editing, functional research, etc. AlpHelix has been engaged in research on membrane proteins and other challenging proteins for many years, with extensive experience in developing stable cell lines. Our professional services can greatly improve the productivity of stable cell lines, thereby speeding research up. We provide a one-stop service from gene synthesis to stable cell line delivery.

1-2weeks 2-3weeks 2weeks 12-20weeks 2-3weeks
Gene synthesis and codon optimization (optional) Generation of expression constructs Transient expression evaluation Stable cell line development Large-scale expression and purification (optional)
Customer provides target protein sequence information or plasmids information · Cloning genes into expression vectors
· vector sequencing
· vector preparation

Deliverables
· Sequencing report (if requested by the customer) 		· Transient expression test
· ELISA or/and WB detection

Deliverables
· Evaluation report 		· Screening for stable cell lines
· Screening for high-expression clones
· Building cell line library

Deliverables
· Expression report
· 2-3 stable clones (optional) 		· 100mg or more protein
· QC analysis

Deliverables
· Purified protein
· Quality control report (COA)

For more information about our technology and services, please contact us.

Contact Us