High-density mammalian transient protein expression

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

See expression data   Expi293 savings calculator

Expi293 Expression System testimonial

In all my years working with transient expression systems the Expi293™ Expression System is the first one to achieve 2.3 grams/L, beating every other HEK293 transient expression system.” - Jelte-Jan Reitsma, Research Associate

Expi293 Expression System videos

Transient Protein Expression—Expi293 Expression System
Expi293: Transient HEK 293 Expression Protocol
Expi293 Expression System - Meet the inventor series

Expi293 Expression System applications

Get 20x greater membrane protein yield

Expi293 MembranePro Expression System
For researchers studying G protein–coupled receptors (GPCRs) and other membrane proteins, the Expi293™ MembranePro™ Expression System delivers enriched, functional membrane proteins with high efficiency, scalability, and reliability.

  • Efficient—20x greater membrane protein yield compared to the adherent system
  • Scalable—easy scale-up without the need for multiple flasks
  • Easy to use—no enzymatic or mechanical dissociation required

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Precise protein expression is all in the fold

For structural biologists, insoluble and misfolded proteins mean starting over. The Gibco™ Expi293™ Expression System is precisely designed with suspension 293 cells, optimized medium and transfection reagent to provide native folding and maximum protein yields. Now, your structural study can get the biological relevance it deserves.

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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.

The Expi293 protocol

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.

Gibco™ Expi293F™ cells are thawed and grown in suspension culture in Gibco™ 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 Gibco™ 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 1).

Expression of Fc-tagged Cripto  
Figure 1. Expression of Fc-tagged Cripto achieves similar volumetric protein yields at 3 different scales.

Application notes

What’s in the Gibco system?

Expi293™ Expression System

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. Each kit includes:

  • 2 vials of frozen Expi293F cells
  • 1 liter of Expi293 Expression Medium
  • 1 ExpiFectamine 293 Transfection Kit, sufficient to transfect 1 liter of culture
  • Opti-MEM™ Reduced Serum Medium
  • An antibody-expressing positive control

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 Invitrogen™ FreeStyle™ 293 Expression System. Expression levels of greater than 1 g/L were achieved for IgG and non-IgG proteins (See Figure 2).

Expi293™ Expression System  
Figure 2. Expression of human IgG and Fc-tagged Cripto achieve expression levels of over 1 g/L in the Expi293 Expression System.

Unique features of the Expi293 Expression System

Expi293 Expression Medium is a chemically defined, serum-free, protein-free medium. Expi293 Expression Medium enables high density cell culture (See Figure 3) and transfection, allowing for significantly higher volumetric protein yields than traditional culture systems.

Expi293™ Expression Medium  
Figure 3. 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 Invitrogen™ FreeStyle™ 293-F 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 4).

Expression of hIgG  
Figure 4. Expression of hIgG, with and without transfection enhancers.

Expi293 System cost comparison chart

  PEI w/ FreeStyle 293 Medium Expi293 System (5x Yield)
Cost of 1L Transfection $548 $1531
Media $458 $893
DNA $90 $90
Tfxn Reagent $0 $548
Culture Volume for Equivalent Yield 1L 200mL
Cost of Transfection $548 $306
Cost of Culture Vessel $59 $18
Total Cost for Same Amount of Protein $607 $324