Abstract
Membrane proteins are essential components in cell membranes and enable cells to communicate with their outside environment and to carry out intracellular signaling. Functional reconstitution of complex membrane proteins using cell-free expression (CFE) systems has been proved to be challenging mainly due to the lack of necessary machinery for proper folding and translocation of nascent membrane proteins and their delivery to the supplied synthetic bilayers. Here, we provide protocols for detergent-free, cell-free reconstitution of functional membrane proteins using HeLa-based CFE system and outline assays for studying their membrane insertion, topology, and their orientation upon incorporation into the supported lipid bilayers or bilayers of giant unilamellar vesicles as well as methods to isolate functional translocated cell-free produced membrane proteins.
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References
Laohakunakorn N, Grasemann L, Lavickova B et al (2020) Bottom-up construction of complex biomolecular systems with cell-free synthetic biology. https://pubmed.ncbi.nlm.nih.gov/32266240/
Wingfield PT (2015) Overview of the purification of recombinant proteins. Curr Protoc Protein Sci 2015:6.1.1–6.1.35
Mancia F, Love J (2010) High-throughput expression and purification of membrane proteins. J Struct Biol 172:85–93
Knol J, Sjollema K, Poolman B (1998) Detergent-mediated reconstitution of membrane proteins. Biochemistry 37:16410–16415
Rigaud J-LL, Lévy D (2003) Reconstitution of membrane proteins into liposomes. Methods Enzymol 372:65–86
Noireaux V, Liu AP (2020) The new age of cell-free biology. Annu Rev Biomed Eng 22:51–77
Chong S (2014) Overview of cell-free protein synthesis: historic landmarks, commercial systems, and expanding applications. Curr Protoc Mol Biol 2014:16.30.1–16.30.11
Lu Y (2017) Cell-free synthetic biology: engineering in an open world. Synth Syst Biotechnol 2(1):23–27
Perez JG, Stark JC, Jewett MC (2016) Cell-free synthetic biology: engineering beyond the cell. Cold Spring Harb Perspect Biol 8:a023853
Khambhati K, Bhattacharjee G, Gohil N et al (2019) Exploring the potential of cell-free protein synthesis for extending the abilities of biological systems. https://pubmed.ncbi.nlm.nih.gov/31681738/
Zemella A, Thoring L, Hoffmeister C et al (2015) Cell-free protein synthesis: pros and cons of prokaryotic and eukaryotic systems. Chembiochem 16:2420–2431
Gregorio NE, Levine MZ, Oza JP (2019) A user’s guide to cell-free protein synthesis. https://pubmed.ncbi.nlm.nih.gov/31164605/
Dondapati SK, Kreir M, Quast RB et al (2014) Membrane assembly of the functional KcsA potassium channel in a vesicle-based eukaryotic cell-free translation system. Biosens Bioelectron 59:174–183
Komiya M, Kato M, Tadaki D et al (2020) Advances in artificial cell membrane systems as a platform for reconstituting ion channels. https://pubmed.ncbi.nlm.nih.gov/31944562/
Demarche S, Sugihara K, Zambelli T et al (2011) Techniques for recording reconstituted ion channels. Analyst 136:1077–1089
Bashirzadeh Y, Liu AP (2019) Encapsulation of the cytoskeleton: towards mimicking the mechanics of a cell. Soft Matter 15:8425–8436
Bashirzadeh Y, Wubshet NH, Liu AP (2020) Confinement geometry tunes fascin-actin bundle structures and consequently the shape of a lipid bilayer vesicle. Front Mol Biosci 7:610277
Bashirzadeh Y, Redford SA, Lorpaiboon C, et al (2021) Actin crosslinker competition and sorting drive emergent GUV size-dependent actin network architecture. Commun Biol 41(4):1–11
Groaz A, Moghimianavval H, Tavella F et al (2020) Engineering spatiotemporal organization and dynamics in synthetic cells. https://pubmed.ncbi.nlm.nih.gov/33219745/
Majumder S, Garamella J, Wang YL et al (2017) Cell-sized mechanosensitive and biosensing compartment programmed with DNA. Chem Commun 53:7349–7352
Majumder S, Willey PT, DeNies MS et al (2019) A synthetic biology platform for the reconstitution and mechanistic dissection of LINC complex assembly. J Cell Sci 132:234153
Neumann S, Pucadyil TJ, Schmid SL (2013) Analyzing membrane remodeling and fission using supported bilayers with excess membrane reservoir. Nat Protoc 8:213–222
Mikami S, Kobayashi T, Imataka H (2010) Cell-free protein synthesis systems with extracts from cultured human cells. Methods Mol Biol 607:43–52
Abkarian M, Loiseau E, Massiera G (2011) Continuous droplet interface crossing encapsulation (cDICE) for high throughput monodisperse vesicle design. Soft Matter 7:4610–4614
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Moghimianavval, H., Hsu, YY., Groaz, A., Liu, A.P. (2022). In Vitro Reconstitution Platforms of Mammalian Cell-Free Expressed Membrane Proteins. In: Karim, A.S., Jewett, M.C. (eds) Cell-Free Gene Expression. Methods in Molecular Biology, vol 2433. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1998-8_6
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DOI: https://doi.org/10.1007/978-1-0716-1998-8_6
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Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1997-1
Online ISBN: 978-1-0716-1998-8
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