Mammalian Expression for Membrane Protein Production Support—Getting Started
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We offer the MembranePro™ Functional Protein Expression System for the expression and display of mammalian cell surface membrane proteins, including G-protein coupled receptors (GPCRs), in an aqueous-soluble format. The system uses virus-like particles (VLPs) to capture lipid raft regions of the cell’s plasma membrane as the VLPs are secreted from the cell. Using this system, it is possible to capture and display endogenous or overexpressed GPCRs and other cell surface membrane proteins in their native context for downstream assays. Since the VLPs are packaged by the cell and secreted into the culture medium, VLPs allow for the isolation of functional membrane proteins by simply decanting and clarifying the culture medium, and isolating the VLPs by precipitation. This represents a substantial savings in time, effort, and required machinery over preparing cell membrane fractions. Because VLPs capture receptor-rich regions of the plasma membrane, your GPCR may also be substantially enriched over crude membrane preparations.
The standard MembranePro™ Functional Protein Expression System is offered in two configurations: The MembranePro™ Functional Protein Expression Kit (Cat. No. A11667), which provides the convenience of a complete kit with all the reagents supplied for 10 reactions, and the MembranePro™ Functional Protein Support Kit, which includes the reagents for functional expression of membrane proteins but does not contain the expression vector cloning module or the 293FT host cells. The MembranePro™ Functional Protein Support Kit is offered in three sizes, allowing 10, 60, or 600 reactions (Cat. Nos. A11668, A11669, A11670, respectively).
For high-yield expression of functional membrane proteins in Exp293F™ cells, we offer the Expi293™ MembranePro™ Expression System (Cat. Nos. A25869, A25870) that combines the scalability and ease of use of Expi293™ and the technology of MembranePro™ to allow an increase of more than 20-fold in membrane protein yield compared to the standard, adherent culture MembranePro™ Functional Protein Expression System. Please see the Application Note for more details.
Note: Expi293F™ cells (Cat. No. A14527) and pEF6 V5 His TOPO® Expression Vector Kit (Cat. No. K961020) are not supplied with the Expi293™ MembranePro™ Expression System and have to be purchased separately as required.
No. However, you can visually tell if MembranePro™ particles formed via a pellet at the bottom of the tube following precipitation. You can also test the function of your MembranePro™ particles via receptor-ligand binding studies.
While other expression vectors and promoters can be tested, we recommend using the pEF6 V5-His TOPO® TA expression vector for optimal yields, as the performance of these kits has been optimized for use with this expression vector containing the EF-1α promoter.
While other cell lines can be tested, we recommend using the Expi293F™ cells for optimal yields, as the performance of the kit has been optimized for use with this cell line (i.e., transfection efficiency using ExpiFectamine™ Transfection Reagent).
Theoretically this is possible, but we have not validated this approach. You should be able to generate MembranePro™ particles using the MembranePro™ Functional Protein Support Kits (Cat. Nos. A11668, A11669, A11670) assuming the cell line has adequate GPCR expression, and functional testing (i.e., receptor-ligand binding studies) with conventional membrane preps from these cell lines was successful. The protein yield for the GPCR will depend on the expression levels from their stable cell line.
The C-terminal His tag in the pEF6 TOPO® TA vector is an appendix and has no function based on its position. It will only be expressed if you create a construct without a stop codon to specifically capture the tag. There is no N-terminal His tag present in the vector.
The GPCRs we produced and tested are ~50 kDa. In theory, there is no limit on the size of the extracellular or transmembrane domains. However, there may be packaging issues if the C-terminal domain is too large.
The gag protein can't be removed unless you dissociate the MembranePro™ particles. Our recommendation is that methods used to isolate membrane proteins should eliminate gag. This system makes analytical quantities of protein, 50–500 μg total protein per reaction, of which a small fraction is your GPCR.
If a ligand-binding chain is transfected alone, there may not be coupling of G-protein. We have demonstrated agonist ligand binding to 5HT1a and muscarinic M1 receptors captured on MembranePro™ particles. Different agonists compete ligand off both muscarinic and 5HT1a receptors in MembranePro™ particles, and they do so in order of affinity, indicating physiologically relevant interaction with the receptors on MembranePro™ particles. In direct binding studies, agonist binds in the low-affinity form (higher Kd), indicating little or no G-protein is coupled to the receptor. However, the binding is still specific and competitive. You may see increased binding by coexpressing the cognate G-protein.
For Research Use Only. Not for use in diagnostic procedures.