Cell-Free Synthesis of Human Endothelin Receptors and Its Application to Ribosome Display

Engineering
G-protein-coupled receptors (GPCRs) for improved stability
or altered function is of great interest, as GPCRs consist of the
largest protein family, are involved in many important signaling pathways,
and thus, are one of the major drug targets. Here, we report the development
of a high-throughput screening method for GPCRs using a reconstituted
in vitro transcription–translation (IVTT) system. Human endothelin
receptor type-B (ETBR), a class A GPCR that binds endothelin-1 (ET-1),
a 21-residue peptide hormone, was synthesized in the presence of nanodisc
(ND) composed of a phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG). The ET-1 binding
of ETBR was significantly reduced or was undetectable when other phospholipids
were used for ND preparation. However, when functional ETBR purified
from Sf9 cells was reconstituted into NDs, ET-1 binding was observed
with two different phospholipids tested, including POPG. These results
suggest that POPG likely supports the folding of ETBR into its functional
form in the IVTT system. Using the same conditions as ETBR, whose
three-dimensional structure has been solved, human endothelin receptor
type-A (ETAR), whose three-dimensional structure remains unsolved,
was also synthesized in its functional form. By adding POPG-ND to
the IVTT system, both ETAR and ETBR were successfully subjected to
ribosome display, a method of in vitro directed evolution that facilitates
the screening of up to 1012 mutants. Finally, using a mock
library, we showed that ribosome display can be applied for gene screening
of ETBR, suggesting that high-throughput screening and directed evolution
of GPCRs is possible in vitro.