Abstract
Cell-free expression allows to synthesize membrane proteins in completely new formats that can relatively easily be customized for particular applications. Amphiphilic superstructures such as micelles, lipomicelles, or nanodiscs can be provided as nano-devices for the solubilization of membrane proteins. Defined empty bilayers in the form of nanodiscs offer native like environments for membrane proteins, supporting functional folding, proper oligomeric assembly as well as stability. Even very difficult and detergent-sensitive membrane proteins can be addressed by the combination of nanodisc technology with efficient cell-free expression systems as the direct co-translational insertion of nascent membrane proteins into supplied preassembled nanodiscs is possible. This chapter provides updated protocols for the synthesis of membrane proteins in presence of preassembled nanodiscs suitable for emerging applications such as screening of lipid effects on membrane protein function and the modulation of oligomeric complex formation.
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References
Hein C, Henrich E, Orbán E et al (2014) Hydrophobic supplements in cell-free systems: designing artificial environments for membrane proteins. Eng Life Sci 14:365–379
Schuler MA, Denisov IG, Sligar SG (2013) Nanodiscs as a new tool to examine lipid-protein interactions. Methods Mol Biol 974:415–433
Henrich E, Hein C, Dötsch V et al (2015) Membrane protein production in Escherichia coli cell-free lysates. FEBS Lett 589:1713–1722
Katzen F, Fletcher JE, Yang JP et al (2008) Insertion of membrane proteins into discoidal membranes using a cell-free protein expression approach. J Proteome Res 7:3535–3542
Roos C, Zocher M, Mueller D et al (2012) Characterization of co-translationally formed nanodisc complexes with small multidrug transporters, proteorhodopsin and with the E. coli MraY translocase. Biochim Biophys Acta 1818:3098–3106
Henrich E, Ma Y, Engels I et al (2016) Lipid requirements for the enzymatic activity of MraY translocases and in vitro reconstitution of the lipid II synthesis pathway. J Biol Chem 291:2535–2546
Rues RB, Dötsch V, Bernhard F (2016) Co-translational formation and pharmacologiocal characterization of beta1-adrenergic receptor/nanodisc complexes with different lipid environments. Biochim Biophys Acta 1858:1306–1316
Orbán E, Proverbio D, Haberstock S et al (2015) Cell-free expression of G-protein-coupled receptors. Methods Mol Biol 1261:171–195
Reckel S, Sobhanifar S, Durst F et al (2010) Strategies for the cell-free expression of membrane proteins. Methods Mol Biol 607:187–212
Acknowledgments
This work was funded by the Collaborative Research Center (SFB) 807 of the German Research Foundation (DFG). Support was further obtained by Instruct, part of the European Strategy Forum on Research Infrastructures (ESFRI).
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Rues, RB., Gräwe, A., Henrich, E., Bernhard, F. (2017). Membrane Protein Production in E. coli Lysates in Presence of Preassembled Nanodiscs. In: Burgess-Brown, N. (eds) Heterologous Gene Expression in E.coli. Methods in Molecular Biology, vol 1586. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6887-9_19
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DOI: https://doi.org/10.1007/978-1-4939-6887-9_19
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