Cell-Free Synthesis Strategies to Probe Co-translational Folding of Proteins Within Lipid Membranes

Nicola J. Harris, Eamonn Reading, Paula J. Booth*

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

1 Citation (Scopus)


In order to comprehend the molecular basis of transmembrane protein biogenesis, methods are required that are capable of investigating the co-translational folding of these hydrophobic proteins. Equally, in artificial cell studies, controllable methods are desirable for in situ synthesis of membrane proteins that then direct reactions in the synthetic cell membrane. Here we describe a method that exploits cell-free expression systems and tunable membrane mimetics to facilitate co-translational studies. Alteration of the lipid bilayer composition improves the efficiency of the folding system. The approach also enables membrane transport proteins to be made and inserted into artificial cell platforms such as droplet interface bilayers. Importantly, this gives a new facet to the droplet networks by enabling specific transport of molecules across the synthetic bilayer against a concentration gradient. This method also includes a protocol to pause and restart translation of membrane proteins at specified positions during their co-translational folding. This stop–start strategy provides an avenue to investigate whether the proteins fold in sequence order, or if the correct fold of N-terminal regions is reliant on the synthesis of downstream residues.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc
Number of pages20
Publication statusPublished - 2022

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029


  • Active transport
  • Artificial cells
  • Cell-free transcription/translation
  • In vitro co-translational folding
  • Lipid bilayers
  • Membrane proteins
  • Translation pausing


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