Creative Biostructure has established an advanced Mempro™ Nanodisc technology platform for a long time. With our sophisticated scientists hammered at Nanodisc research for years, we can provide customized Mempro™ cell-free system for membrane proteins reconstituted in Nanodisc services.
Production of membrane proteins (MPs) in a folded structure is a crucial precondition for their functional and structural researches. However, in contrast to water-soluble globular proteins, MPs researches are indeed difficult due to the demand for a membrane-like state to stabilize a right spatial structure and to keep a functional activity of IMPs in solution. Cell-free (CF) expression system has turned into a common technique for the production of membrane proteins, either from prokaryotic or eukaryotic origin. By using CF expression systems, several proteins has been produced in functionally folded state, such as G-protein-coupled receptors, porins, transporters and so on. The continuous exchange cell-free (CECF) is generally used for preparative scale production of MPs.
Figure 1. Scheme for the preparation of bR loaded nanodiscs. (PloS one 2013)
In comparison with common cell-based expression, CF expression system has many advantages, for instance, it enables to synthesis of toxic proteins, observably simplify synthesis of the selectively labeled proteins for their structural and biochemical studies, allow a direct accretion of the agents or co-factors to the reaction mixture, which accelerate the production of the native structure of the synthesized protein and promote its stability in solution. The ingredients of the traditional membrane mimicking media can be put in to the reaction mixture to facilitate membrane protein production in a soluble form, which includes lipid/detergent mixtures (bicelles), liposomes, fluorinated surfactants and amphipols.
Main points of the efficient solubilization of CF-expressed membrane proteins are extremely concentrated and homogenous nanodisc stock solutions. Nanodisc enables to be filled with a great number of different lipids, and the chosen type of lipid has important effects on the solubilization efficacy of individual membrane proteins. Creative Biostructure has developed an approach for the co-translational association/insertion of CF-expressed membrane proteins with nanodisc. With this approach Nanodisc is sufficient tolerated by CF systems and generally do not have repressive effects on the expression efficiency.
Creative Biostructure provides other various Mempro™ membrane proteins reconstituted in Nanodisc services. Please feel free to contact us for a detailed quote.
Roos C, Kai L, Haberstock S, et al. High-level Cell-free production of membrane proteins with Nanodiscs[J]. Cell-Free Protein Synthesis: Methods and Protocols, 2014: 109-130.
Lyukmanova E N, Shenkarev Z O, Khabibullina N F, et al. Lipid–protein nanodiscs for cell-free production of integral membrane proteins in a soluble and folded state: comparison with detergent micelles, bicelles and liposomes[J]. Biochimica et Biophysica Acta (BBA)-Biomembranes, 2012, 1818(3): 349-358.
Pavlidou M, Hänel K, Möckel L, et al. Nanodiscs allow phage display selection for ligands to non-linear epitopes on membrane proteins[J]. PloS one, 2013, 8(9): e72272.