Article

Co-translational association of cell-free expressed membrane proteins with supplied lipid bilayers.

Centre for Biomolecular Magnetic Resonance, Institute for Biophysical Chemistry, Goethe-University of Frankfurt/Main , Frankfurt/Main , Germany.
Molecular Membrane Biology (Impact Factor: 1.73). 06/2012; DOI: 10.3109/09687688.2012.693212
Source: PubMed

ABSTRACT Abstract Routine strategies for the cell-free production of membrane proteins in the presence of detergent micelles and for their efficient co-translational solubilization have been developed. Alternatively, the expression in the presence of rationally designed lipid bilayers becomes interesting in particular for biochemical studies. The synthesized membrane proteins would be directed into a more native-like environment and cell-free expression of transporters, channels or other membrane proteins in the presence of supplied artificial membranes could allow their subsequent functional analysis without any exposure to detergents. In addition, lipid-dependent effects on activity and stability of membrane proteins could systematically be studied. However, in contrast to the generally efficient detergent solubilization, the successful stabilization of membrane proteins with artificial membranes appears to be more difficult. A number of strategies have therefore been explored in order to optimize the co-translational association of membrane proteins with different forms of supplied lipid bilayers including liposomes, bicelles, microsomes or nanodiscs. In this review, we have compiled the current state-of-the-art of this technology and we summarize parameters which have been indicated as important for the co-translational association of cell-free synthesized membrane proteins with supplied membranes.

4 Bookmarks
 · 
284 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Liposome display is a novel method for in vitro selection and directed evolution of membrane proteins. In this approach, membrane proteins of interest are displayed on liposome membranes through translation from a single DNA molecule by using an encapsulated cell-free translation system. The liposomes are probed with a fluorescence indicator that senses membrane protein activity and selected using a fluorescence-activated cell sorting (FACS) instrument. Consequently, DNA encoding a protein with a desired function can be obtained. By implementing this protocol, researchers can process a DNA library of 10(7) different mutants. A single round of the selection procedure requires 24 h for completion, and multiple iterations of this technique, which take 1-5 weeks, enable the isolation of a desired gene. As this protocol is conducted entirely in vitro, it enables the engineering of various proteins, including pore-forming proteins, transporters and receptors. As a useful example of the approach, here we detail a procedure for the in vitro evolution of α-hemolysin from Staphylococcus aureus for its pore-forming activity.
    Nature Protocols 07/2014; 9(7):1578-1591. · 7.78 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Membrane proteins are key elements in cell physiology and drug targeting, but getting a high-resolution structure by crystallographic means is still enormously challenging. Novel strategies are in big demand to facilitate the structure determination process that will ultimately hasten the day when sequence information alone can provide a three-dimensional model. Cell-free or in vitro expression enables rapid access to large quantities of high-quality membrane proteins suitable for an array of applications. Despite its impressive efficiency, to date only two membrane proteins produced by the in vitro approach have yielded crystal structures. Here, we have analysed synergies of cell-free expression and crystallisation in lipid mesophases for generating an X-ray structure of the integral membrane enzyme diacylglycerol kinase to 2.28-Å resolution. The quality of cellular and cell-free-expressed kinase samples has been evaluated systematically by comparing (1) spectroscopic properties, (2) purity and oligomer formation, (3) lipid content and (4) functionality. DgkA is the first membrane enzyme crystallised based on cell-free expression. The study provides a basic standard for the crystallisation of cell-free-expressed membrane proteins and the methods detailed here should prove generally useful and contribute to accelerating the pace at which membrane protein structures are solved.
    Cellular and Molecular Life Sciences CMLS 07/2014; · 5.86 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Membrane proteins are of central interest for the pharmaceutical industry but their production is usually a challenging task. The complex folding mechanisms of newly synthesized membrane proteins often require interactions with specific compounds for improved stability. Conditions for the production of high quality samples are therefore difficult to predict and frequently cannot be provided in conventional protein expression platforms. Cell-free biosynthetic systems allow, in contrast to living cells, non-restricted access to the protein production machinery. Reaction conditions can be adjusted according to particular requirements and they can be modified by supplementing single additives or even cocktails of compounds. These options have initiated completely new research fields for the co-translational stabilization and folding of membrane proteins in artificial environments. Based on established and efficient cell-free production protocols, a recent focus was to explore and to define suitable supplements for cell-free expression reactions that are useful for the generation of high quality membrane protein samples. Besides classical detergents and lipids, a variety of new compounds with interesting properties have been discovered and synthesized. We compile the currently available toolbox for membrane protein solubilization in cell-free systems and we summarize new developments and perspectives for the directed modulation of cell-free biosynthetic environments. This article is protected by copyright. All rights reserved
    Engineering in Life Sciences 02/2014; 14(4). · 1.89 Impact Factor

Full-text

Download
117 Downloads
Available from
Jun 1, 2014