ATP
A
K
Application Note
Active, Monomeric
ß1AR
in <72h: Cell-Free GPCR
Synthesis via Multiplexed Nanodisc Screening
Thomas Guilliam, Elena Rahmani, and Ruben Tomás
Nuclera Ltd, Cambridge UK
Introduction
G protein-coupled receptors (GPCRs) are targets for ~35% of approved drugs, but structurally
characterized GPCRs remain disproportionately underrepresented relative to the size of the receptor
family.1,2 A major contributor is the difficulty of producing stable, functional receptor in detergents:
.1,2
solubilization can strip stabilizing lipids, increase aggregation, and reduce ligand-binding competency. As
a result, teams often spend weeks iterating detergent and construct conditions before obtaining assay
or
structure-grade material.
Cell-free protein synthesis (CFPS) offers a powerful alternative by bypassing cellular toxicity and enabling
the co-translational insertion of membrane proteins. By including pre-assembled membrane scaffold
protein (MSP)-lipid nanodiscs directly in the translation mixture, nascent GPCRs insert spontaneously
into a native-like lipid bilayer. This detergent-free, “stabilize as you synthesize” approach preserves key
lipid–protein interactions and supports rapid exploration of a tunable design space (scaffold size and lipid
chemistry) to maximize yield and functional folding.
Setup cell-free
Co-translational insertion
Stable and
expression
into nanodisc
active GPCR
A
V
K
Polymerase
A
G
G
L
V
K
Polymerase
M
ATP
Cell-free
Cell-free
core reagent
M
Mg2+
DNA
Pre-assembled
Template
nanodiscs
Ribosome
DNA
T7-RNA
Polymerase
mRNA
Figure 1. CFPS enables the co-translational insertion of GPCRs into defined lipid environments during translation.
G
L
V
K
P
L
V
K
P
G
