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

TY - CHAP

T1 - Cell-Free Synthesis Strategies to Probe Co-translational Folding of Proteins Within Lipid Membranes

AU - Harris, Nicola J.

AU - Reading, Eamonn

AU - Booth, Paula J.

N1 - Funding Information: We acknowledge funding from the European Research Council, ERC Advanced grant 294342 and Wellcome Trust Investigator Award 214259/Z/18/Z to PJB., and funding from a BBSRC Future Leader Fellowship [BB/N011201/1] and a UKRI Future Leaders Fellowship [MR/S015426/1] to E.R. Publisher Copyright: © 2022, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

PY - 2022

Y1 - 2022

N2 - 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.

AB - 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.

KW - Active transport

KW - Artificial cells

KW - Cell-free transcription/translation

KW - In vitro co-translational folding

KW - Lipid bilayers

KW - Membrane proteins

KW - Translation pausing

UR - http://www.scopus.com/inward/record.url?scp=85122803522&partnerID=8YFLogxK

U2 - 10.1007/978-1-0716-1998-8_17

DO - 10.1007/978-1-0716-1998-8_17

M3 - Chapter

AN - SCOPUS:85122803522

T3 - Methods in Molecular Biology

SP - 273

EP - 292

BT - Methods in Molecular Biology

PB - Humana Press Inc

ER -