High-Density Mammalian Transient Protein Expression

  • Higher transfection efficiency
  • Up to 10x greater protein yield
  • Improved cell viability
  • Smaller culture volumes

Expi293 Application Notes

Quote From Researcher

The Expi293™ System gives you the benefit of a stable cell system in much less time.  It’s easy to produce 3–5 times more protein with much less effort.

The Expi293™ Expression System is a major advancement in transient expression technology for rapid and ultra high-yield protein production in mammalian cells. It is based on high density culture of Expi293F™ cells in Expi293™ Expression Medium. Transient expression is powered by the new cationic lipid-based ExpiFectamine™ 293 transfection reagent in combination with optimized transfection enhancers designed to work specifically with this transfection reagent. All components work in concert to generate 2- to 10-fold higher protein yields than conventional culture systems such as the FreeStyle™ 293 Expression System.  Expression levels of greater than 1 g/L were achieved for IgG and non-IgG proteins (See Figure 1).

Learn about Expi293™ system on the ambr™ platform for automated protein expression

Expi293™ Expression System  
Figure 1. Expression of human IgG and Fc-tagged Cripto achieve expression levels of over 1g/L in the Expi293™ Expression System.

What’s in the System?

Expi293™ Expression SystemThe components included in the kit are:

  • 2 vials of frozen Expi293F™ cells
  • 1 liter of Expi293™ Expression Medium
  • 1 ExpiFectamine™ 293 Transfection Kit, sufficient to transfect 1 liter of culture
  •  OptiMEM® Reduced Serum Medium
  • An Antibody-Expressing Positive control

The ExpiFectamine™ 293 Reagent and ExpiFectamine™ 293 Transfection Enhancers 1 and 2 are sold together as the ExpiFectamine™ 293 Transfection kit. All other components of the system are available separately.

What Makes Expi293™ Expression System Unique?

Expi293™ Expression Medium is a chemically defined, serum-free, protein-free medium. Expi293™ Expression Medium enables high density cell culture (See Figure 2) and transfection, allowing for significantly higher volumetric protein yields than traditional culture systems.

Expi293™ Expression Medium  
Figure 2. Cell viability and viable cell density over time for cells grown in Expi293™ Expression Medium vs. other 293 culture media.

Expi293F™ cells are specifically adapted to exhibit higher pg/cell/day productivity than typical 293 cells and produce up to 1.7x more protein than Freestyle™ 293F cell when used in the Expi293™ Expression System.

ExpiFectamine™ 293 transfection reagent is capable of high-efficiency transfection of high-density 293 cultures, and is coupled with transfection enhancers that further boost transfection performance and expression levels. (See Figure 3).

Expression of hIgG  
Figure 3. Expression of hIgG, with and without transfection enhancers.

How Does the Kit Work?

The Expi293™ Expression System contains all the components needed to easily produce high levels of recombinant protein in your own laboratory.  The only additional component required is a mammalian expression vector containing the DNA for your protein of interest.

Expi293F™ cells are thawed and grown in suspension culture in Expi293™ Expression Medium. Cells are typically grown in flasks on a shaker platform inside a CO2 cell culture incubator or in bioreactors. The cells are transfected using ExpiFectamine™ 293 Transfection Reagent. Transfection enhancers 1 and 2 are then added to the culture 16-18 hours post-transfection.  There is no need to change culture medium or otherwise supplement the cultures during protein production.  Protein harvest can be performed from 2 to 7 days post-transfection, depending on the expression profiles of the protein of interest. For expression of recombinant antibodies, maximum yields are often seen from 5 to 7 days post-transfection.

The system is highly scalable and should produce similar volumetric protein yields  in transfection formats ranging from 1 mL cultures in a 24-well plate up to 1L cultures in shake flasks (See Figure 4).

Expression of Fc-tagged Cripto  
Figure 4. Expression of Fc-tagged Cripto achieves similar volumetric protein yields at 3 different scales.

The Expi293™ Expression System is a rapid method of producing mg to gram quantities of recombinant protein in mammalian cells. For the expression of human or other mammalian proteins, expression in the human-derived 293 cells offers the benefit of more native folding and post-translational modifications, such as glycosylation, when compared to expression systems based on hosts such as E. Coli, yeast, or insect cells.

Expi293™ System Cost Comparison Chart


PEI with FreeStyle™ Expression Medium
(1 L culture)

Expi293™ System
(1 L culture)

Expi293™ System
(1 L culture)

Total cost $548 $1,531 $1,531
Media cost $458 $893 $893
Transfection reagent cost $ 0 $548 $548
Estimated plasmid cost $90 $90 $90
Protein yield differential* 1x 3x 5x
Culture volume for equivalent protein yield 1 L 335 mL 200 mL
Approximate culture vessel cost† $59 $26 $18
Total cost for equivalent protein yield $607 $536 $324

*Protein yields in each culture are expressed as normalized values, based on the amount obtained using FreeStyle™ Expression Medium supplemented with PEI in 1 L of culture.

Disposable Erlenmeyer shake flasks, Cost/unit:
500 mL, non-baffled, vent cap - $18.00
1000 mL, non-baffled, vent cap - $25.81
3000 mL, Fernbach-style, non-baffled, vent cap - $58.51