Article

Insertion of membrane proteins into discoidal membranes using a cell-free protein expression approach

Invitrogen Corporation, Carlsbad, CA 92008, USA.
Journal of Proteome Research (Impact Factor: 5). 07/2008; 7(8):3535-42. DOI: 10.1021/pr800265f
Source: PubMed

ABSTRACT We report a cell-free approach for expressing and inserting integral membrane proteins into water-soluble particles composed of discoidal apolipoprotein-lipid bilayers. Proteins are inserted into the particles, circumventing the need of extracting and reconstituting the product into membrane vesicles. Moreover, the planar nature of the membrane support makes the protein freely accessible from both sides of the lipid bilayer. Complexes are successfully purified by means of the apoplipoprotein component or by the carrier protein. The method significantly enhances the solubility of a variety of membrane proteins with different functional roles and topologies. Analytical assays for a subset of model membrane proteins indicate that proteins are correctly folded and active. The approach provides a platform amenable to high-throughput structural and functional characterization of a variety of traditionally intractable drug targets.

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Available from: Jenny Cappuccio, Aug 30, 2015
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    • "However, the addition of bicelles to NLPs opens them, and the NLP lipids and proteins transfer to bicelles [5]. Membrane proteins can be inserted into NLPs by cell-free protein synthesis [6]. By manipulating steric and stoichiometric factors in cell-free protein synthesis, it should be possible to prepare predominantly "
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    • "Recent advances in heterologous cell-free (CF) expression systems, however, are presenting exciting new approaches for the preparative scale (milligram quantity) production of MPs and their cotranslational reconstitution into membrane mimetics (Junge et al., 2011). Several groups have successfully used CF systems to incorporate MPs into nanodiscs that were pre-assembled and added to the translation reaction (Katzen et al., 2008; Lyukmanova et al., 2012; Yang, Cirico, Katzen, Peterson, & Kudlicki, 2011) or to co-express the target protein and MSP in the presence of phospholipids, thereby enabling the in situ assembly of the MP-containing nanodisc (Cappuccio et al., 2008). "
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    • "Over the last decade, cell-free methods have proven themselves as a valuable platform allowing the synthesis of many different protein classes including membrane proteins [1], [2], [3], [4], [5], [6], [7], proteins with posttranslational modifications [8], [9], [10], [11], [12], [13], [14] and even toxic proteins [15], [16], [17]. Many problematic issues connected with a cell-based expression of proteins, such as protein insolubility and toxicity, can be circumvented by the use of tailor-made cell-free expression systems. "
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