Get 20x greater membrane protein yield!

The high-yield Expi293™ transient expression system meets the challenge of difficult to express membrane proteins. Membrane proteins are crucial components in many cellular processes and are key targets for both basic research and drug discovery. These essential proteins, such as GPCRs, transporters, adhesion molecules, ion channels and viral envelope proteins, are often difficult to obtain in the needed quantity and quality for study. There is a clear need for expression systems that can provide high amounts of structurally intact, properly oriented and functional membrane proteins. To meet this need, we have combined components from the MembranePro™ Functional Protein Expression system (catalog #A11667) with the mammalian-cell based Expi293™ high-yield transient expression system.

Rapid and ultra high-yield protein production in mammalian cells

The Expi293™ Expression system is a rapid, scalable system that delivers the highest protein yields of any current transient expression method. Typical Expi293 yields are 2- to 10-fold higher than other options. We have observed expression levels of greater than 1 gram per liter of culture for some proteins (Figure 1).

Figure 1. Expi293 Protein Expression. Expression of human erythropoietin (EPO), crypto and a human IgG in the FreeStyle™ 293 system (light columns) compared to expression in Expi293™ (dark columns). Expression was performed in 1ml, 30mL and 1 liter cultures. Protein was harvested at day 5 to day 7 post-transfection. All yields are given as mg of protein per L of culture transfected (mg/L).


Expi293 MembranePro Expression System provides superior yields of difficult to express membrane proteins

The key features that bring about the superior protein yields of the Expi293 system are the enriched Expi293™ Expression Medium, which supports high density culture of superior productivity Expi293F™ cells, and transient expression powered by the novel ExpiFectamine™ 293 transfection reagent and enhancers, which are designed to work specifically under high density culture conditions. All components work in concert to additively increase expression.

MembranePro™ technology delivers overexpressed membrane proteins on virus-like particles (VLPs) that self-assemble and bud off the cell surface. This enables these proteins to be displayed in their native context in a highly enriched nano-lipoparticle. The VLPs are easily collected from the culture medium, providing a workflow that is much less labor intensive than preparation of traditional membrane fractions (Figure 2).

  Figure 2. Expi293-MembranePRO Workflow. Expi293F™ cells in Expi293™ expression medium are transfected with a plasmid expressing the membrane protein of interest and the MembranePRO™ reagent according to the recommended protocol using Expifectamine 293 reagent. 48 hours or more post-transfection, the VLPs containing the membrane protein of interest can be harvested by centrifuging the culture medium to remove cells and cell debris, then precipitating the VLPs from the clarified culture supernatant and resuspending them in PBS.

By combining the capabilities of the high expression Expi293™ system and MembranePRO™ technology researchers can obtain greater than 20-fold increases in membrane protein yield compared to traditional adherent culture protein expression and up to 30-fold enrichment of the membrane protein on the surface of the VLPs compared to membrane fractions (Figure 3). These two technologies work together to clearly provide a solution to meet the challenge of expressing membrane proteins.

  Membrane Fraction MembranePRO VLP Expi293-MembranePRO VLP
Protein Yield (µg/µL)
-- 0.063 2.79
Fold increase in yield
-- 1
44
Binding Constant (Kd)
0.708nM 0.93nM
1.06nM
Specific Bound Bmax
(pg ligand/mg protein)
7.184 39.68
30.08
Fold enrichment of receptor on VLP
-- 5.5 4.2

Figure 3. Expi293-MembranePRO expression of Beta2 Adrenergic Receptor. Comparison of the production of the GPCR Beta2 adrenergic receptor (B2Ar) expressed by traditional transfection of adherent 293FT cells and harvested as a membrane fraction, or expressed by the standard MembranePRO protocol using adherent 293FT, or by the Exp293-MembranePRO method. VLP yields were determined by standard Bradford assays. Fold enrichment of the GPCR on the VLPs compared to membrane fractions were calculated based on comparison of specific bound Bmax values. Expression of the Seratonin 5HT1a GPCR using MembranePRO technology resulted in a 30-fold enrichment of the receptor on VLPs (data not shown).