Assembled nanodiscs are available as part of a collaboration with the Institute of Biophysics at the University of Frankfurt. They are conveniently aliquoted in 50 µL solutions of 0.5 mM concentration. End concentrations in the cell-free reaction range from 20-80 µM. Bacterial and eucaryotic membrane proteins can be expressed (Fig 1 and 2), activity may depend on the lipid composition of the assembled nanodisc. Alternatively, nanodiscs can be assembled from the pre-aliquoted components provided in our Nanodisc Assembly kits
Please note that assembled nanodiscs are intended for use in cell-free expression reactions only. They are not recommended for the reconstitution of membrane proteins already solubilized in detergent, or for the solubilization of proteins from membranes.
Available assembled nanodisc variations:
DMPC and POPC as eukaryotic phospholipids
DMPG as prokaryotic phospholipid
MSP1D1 (wild-type), MSP1D1dH5, MSP2N2 and MSPE3D1 scaffold proteins, both his-tagged and untagged
Nanodisc MSP1D1-His_DMPC was successfully used
in the following publication:
Guo, S.. et al. Expressing Biologically Active Membrane Proteins in a Cell-Free Transcription-Translation Platform.
bioRxiv preprint Jan 30, 2017; doi: http://dx.doi.org/10.1101/104455
Fig. 1: Stability and specific activity of E. coli MraY translocase in different lipid environments. The protein was cell-free synthesized in E. coli lysates in presence of pre-assembled MSP1E3D1 nanodiscs containing the indicated lipids. The topology of the membrane integrated MraY enzyme is indicated in the inset. MraY catalyses the lipid-I precursor formation of bacterial cell wall biosynthesis. The figure shows the lipid-I formation in nmol per µg catalyzed by the corresponding MraY/nanodisc samples (Roos et al., (2012) BBA 1818, 3098-3106). The activity of the enzyme strongly depends on anionic phosphatidylglycerol type lipids and was best with the shorter myristoyl fatty acid chains (DMPG). Data kindly provided by Frank Bernhard, University Frankfurt.
Fig. 2. Differential ligand binding activity of the human endothelin A (ETA) and endothelin B (ETB) receptors in MSP1E3D1 (DMPC) nanodiscs. The G protein-coupled receptors (GPCRs) ETA and ETB were cell-free synthesized in E. coli lysates in presence of pre-assembled MSP1E3D1 nanodiscs containing the lipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). The resulting receptor/nanodisc samples were analyzed for their characteristic binding affinities to four different peptide ligands by SPR (Proverbio et al., (2013) BBA 1828, 2182-2192). Both GPCRs have high affinity to the natural ligand ET-1 while the affinities to the other three ligands is receptor specific. The characteristic differential ligand binding profile of the cell-free synthesized GPCR/nanodisc samples was obtained and in good agreement with data from the literature. Data kindly provided by Frank Bernhard, University Frankfurt.