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Combination of Nanodiscs and Cell-Free Expressed Membrane Proteins

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Creative Biolabs provides combination of expression of membrane proteins by Mempro™ cell-free (CF) system with nanodisc technology employing discoidal membrane structures of 10–20 nm in diameter formed by a lipid bilayer core and surrounded by the membrane scaffold protein. Nanodiscs are widely used for solubilizing seven-transmembrane GPCRs in a soluble and functional state due to this technology can offer near-native membrane environment.

Creative Biolabs can obtain various membrane proteins using Mempro™ cell-free protein production services. Considering the potential drawback of detergents, a novel method, represents currently the most efficient production technique for membrane proteins, is engineered. After completion of protein expression by CF system, dialysis membranes with a cut-off of 12-14 kDa can be used to separate the reaction mixture (RM) from the feeding mixture (FM). Then, cell-free expressed membrane proteins are collected as supernatant [CF expression system in presence of lipids (L-CF) and nanodiscs] by centrifugation.

Lipids for L-CF expression are Aso-PC, DMPC, DOPC, heart total lipid extract, and E. coli polar lipid mixture. The lipids are solubilized in chloroform, evaporated under vacuum to a thin film. The lipid film is resuspended in liposme buffer, then directly added into the RM. Pre-preparation of Nanodiscs is assembled with DMPC, POPC, DMPG, Aso-PC, Aso-PC + 5% cholesterol hemisuccinate and brain total extract. The lipids are mixed with the purified MSPs in the presence of 0.1% DPC. Formation of nanodiscs are performed by dialysis. Final step, pre-formed nanodiscs are directly added into the RM, then the mixtureof synthesized membrane proteins and liposomes will be integrated into the nanodiscs (Figure 1). One significant advantage in this method is not required for detergents, minimizing possible protein denaturation.

MCombination of Nanodiscs and Cell-Free Expressed Membrane Proteins Figure 1. Schematic of reconstitution of cell-free expressed membrane proteins into nanodiscs.

As the membran proteins from nanodiscs reconstitutiom are expressed by Mempro cell-free protein prodution services, which enable introduction of protein modifications (such as biotinylation or isotope labelling). Moreover, ligand binding assays and Surface plasmon resonance (SPR) can be performed using this reconstitution method.

Creative Biolabs can provide other membrane protein production services. Please feel free to contact us for a detailed quote.

References:
A. Leitz et al. (2006). Functional reconstitution of β2-adrenergic receptors utilizing self-assembling Nanodisc technology. BioTechniques. 40(5): 601-606.
A. Robinson et al. Production of Membrane Proteins.
D. Proverbio, et al. (2013). Functional properties of cell-free expressed human endothelin A and endothelin B receptors in artifical membrane environments. Biochim.Biophys. Acta., 1828(9):2182-92.
R. M. Bill, et al. (2011). Surfactant-free purification of membrane proteins with intact native membrane environment. Biochem. Soc. Trans. 39: 813-818.




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