Home > Protein Expression > Mammalian Expression
Page Contents
1. Background and Advantages
2. Service Overview
3. Service Details
Mammalian Expression Systems and Services
Background and Advantages
Transient mammalian protein expression is the expression of a gene for a limited time after introduction of DNA into mammalian cells. A gene encoding the protein of interest is introduced on a plasmid surrounded by the appropriate expression signals such as promoters, terminators and poly(A) signals. Genetic manipulations are carried out during gene synthesis and multiple genes can be expressed simultaneously. We routinely perform transient expression using the following cell lines:
HEK293-S
CHO-S
ExpiCHO*
* mtibio is licensed by Thermo Fisher Scientific to produce proteins in this proprietary expression system. The ExpiCHO system produces yields in excess of 30 times greater than those produced in conventional CHO-S systems.
The advantage of transient mammalian expression include:
Improved solubility due to correct (eukaryotic) protein refolding.
Expression of functional proteins/enzymes/receptors.
Compatibility with all known purification tags.
Endotoxin-free expression of desired protein(s).
Expression of high levels of secreted proteins.
Post-translational modifications such as N- and O-glycosylation and phosphorylation.
Simultaneous expression of multiple proteins / protein complexes.
Accommodation of large genes.
We offer a full range of custom mammalian expression and purification services. To get started on a project or for more information, please contact us.
Mammalian Expression Service Overview
| Step | Description | Deliverable | Timeline |
|---|---|---|---|
| Expression Construct Design | Options: 1. Codon optimization followed by de novo gene synthesis and subcloning into a mammalian expression vector. 2. You suply the completed transfer construct. *Codon optimization for the selected mammalian cell line can increase expression levels. |
Sequence-verified construct | 1-3 weeks |
| DNA Production | The expression construct carried on a DNA plasmid is amplified in E. coli and transfection-grade DNA is prepared. Transfection-grade DNA is extra pure, low-endotoxin, and sterile. | Milligram quantities of expression construct/plasmid DNA | 1 week |
| Transfection | DNA is combined with mamallian cells using special cationic detergents ("fectins") casuing the cells to uptake DNA. Over the next few days, the cells will express the transfected gene and produce the desired protein. Proteins (A) may remain within the cell (i.e., intrinsically expressed) or (B) may be secreted into media if they possess secretion signals. |
Mammalian cells expressing the target protein | 1 week |
| Expression Testing | A small-scale transfection is performed to verify expression and estimate yields for scale-up purposes. Samples of the transfected cells are analyzed for protein yield at various times after transfection. | Experimental report with yield estimates and images of SDS-gels, etc. | 1-2 weeks |
| Protein Production | Mammalian cell cultures are expanded to the appropriate volume to produce the desired yield. Expanded cultures are transfected with DNA then harvested at the optimal time followed by purification of the target protein. | Cell pellets or media | 1-2 weeks |
Transient Mammalian Protein Expression Service Details
We offer a full range of mammalian transfection and protein purification services.
Expression Construct Design
The gene encoding your target protein will be designed to optimize expression in either HEK293 or CHO cell lines. The de novo synthesized gene will be subcloned into an expression vector. Most mammalian expression vectors use the very strong CMV immediate early promoter to drive expression of the transfected gene followed by a poly(A) signal.
DNA Production
The production of high-purity DNA is crucial to optimal transfection and protein expression. We routinely use alkaline lysis followed by endotoxin removal and DNA affinity purification to produce ultra-clean plasmid DNA. This highly purified DNA is then sterilized in preparation for transfection.
Transfection & Expression Testing: Verification and Optimization
A small-scale transfection is performed to verify expression of the target protein and to determine the optimal time for harvest. HEK293 and CHO-S test transfections will analyze culture samples up to 10 days post-transfection whereas ExpiCHO sampling may extend out to 14 days post-transfection. Samples can be either cells (for intrinsic proteins) or media (for secreted proteins).
Time points will be processed for your protein by the most cost-effective method to determine the levels of expression in terms of milligrams per liter. Those methods of analysis include:
Direct SDS-gel analysis of cell lysates or media;
Affinity pull-downs of lysates or media;
Western blot using target-specific antibodies.
Protein Production
Once protein expression has been verified with optimal harvest times, mammalian cell cultures are expanded to accommodate large scale production. Most mammalian production runs are under 10 liters, but production runs can be scaled up to 20 liters. Cells or media are processed at the end of the expression period in preparation for protein purification.
Please contact us for a quote or additional information or take a look at our other custom service offerings for your protein production, expression, and purification needs.