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: 4.25). 07/2008; 7(8):3535-42. DOI: 10.1021/pr800265f
Source: PubMed


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.

Download full-text


Available from: Jenny Cappuccio
  • Source
    • "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 "
    [Show abstract] [Hide abstract]
    ABSTRACT: Nanolipoprotein particles (NLPs), also known as nanodiscs, are lipid bilayers bounded by apolipoprotein. Lipids and membrane proteins cannot exchange between NLPs. However, addition of bicelles opens NLPs and transfers their contents to bicelles, which freely exchange lipids and proteins. NLP-bicelle interactions may provide a new method for studying membrane protein oligomerization. The interaction mechanism was investigated by stopped flow fluorometry. NLP lipids included fluorescence resonance energy transfer donors and acceptors. NLPs were mixed with a 200-fold molar excess of dihexanoyl phosphatidylcholine (DHPC)/dimyristoyl phosphatidylcholine bicelles, and the rate of lipid transfer was monitored by the appearance of dequenched lipid donor fluorescence. The observed pseudo-first-order rate constant was surprisingly small. NLPs did not react with DHPC alone below its critical micelle concentration (cmc). Above the cmc, the reaction was complete within the instrument dead time. Thus, the rate-limiting step is not the reaction of NLPs with DHPC monomers or micelles. Added MSP1E3D1 had no effect on the rate, ruling out free apolipoprotein involvement. The NLP-bicelle mixing rate showed a strong temperature dependence (activation energy = 28 kcal/mol). Near or below the DMPC phase transition temperature, the kinetics were sigmoidal. Models are proposed for the NLP- bicelle mixing, including one involving fusion pores.
    Full-text · Article · Jan 2015 · Biophysical Chemistry
  • Source
    • "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). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Within the last decade, nanoscale lipid bilayers have emerged as powerful experimental systems in the analysis of membrane proteins (MPs) for both basic and applied research. These discoidal lipid lamellae are stabilized by annuli of specially engineered amphipathic polypeptides (nanodiscs) or polymers (SMALPs/Lipodisqs®). As biomembrane mimetics, they are well suited for the reconstitution of MPs within a controlled lipid environment. Moreover, because they are water-soluble, they are amenable to solution-based biochemical and biophysical experimentation. Hence, due to their solubility, size, stability, and monodispersity, nanoscale lipid bilayers offer technical advantages over more traditional MP analytic approaches such as detergent solubilization and reconstitution into lipid vesicles. In this article, we review some of the most recent advances in the synthesis of polypeptide- and polymer-bound nanoscale lipid bilayers and their application in the study of MP structure and function.
    Full-text · Article · Jul 2014 · Biotechnology & genetic engineering reviews
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: In this study, we present a novel technique for the synthesis of complex prokaryotic and eukaryotic proteins by using a continuous-exchange cell-free (CECF) protein synthesis system based on extracts from cultured insect cells. Our approach consists of two basic elements: First, protein synthesis is performed in insect cell lysates which harbor endogenous microsomal vesicles, enabling a translocation of de novo synthesized target proteins into the lumen of the insect vesicles or, in the case of membrane proteins, their embedding into a natural membrane scaffold. Second, cell-free reactions are performed in a two chamber dialysis device for 48 h. The combination of the eukaryotic cell-free translation system based on insect cell extracts and the CECF translation system results in significantly prolonged reaction life times and increased protein yields compared to conventional batch reactions. In this context, we demonstrate the synthesis of various representative model proteins, among them cytosolic proteins, pharmacological relevant membrane proteins and glycosylated proteins in an endotoxin-free environment. Furthermore, the cell-free system used in this study is well-suited for the synthesis of biologically active tissue-type-plasminogen activator, a complex eukaryotic protein harboring multiple disulfide bonds.
    Full-text · Article · May 2014 · PLoS ONE
Show more