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In Vitro Reconstitution Platforms of Mammalian Cell-Free Expressed Membrane Proteins

Part of the Methods in Molecular Biology book series (MIMB,volume 2433)

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.

Key words

  • Cell-free expression
  • Membrane proteins
  • In vitro reconstitution
  • HeLa-based cell-free expression
  • SUN
  • Linker of nucleoskeleton and cytoskeleton complex
  • Encapsulation
  • Giant unilamellar vesicle

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  • DOI: 10.1007/978-1-0716-1998-8_6
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Correspondence to Allen P. Liu .

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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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