Overexpression of relaxin family peptide receptor 3 in Escherichia coli and characterization of its ligand binding properties

[Display omitted] •RXFP3 is an A-class GPCR using neuropeptide relaxin-3 as its cognate ligand.•We overexpressed sMBP-RXFP3 fusion protein using E. coli expression system.•The E. coli-expressed sMBP-RXFP3 retained ligand binding function.•Its ligand binding parameters were affected by lipids of E. coli cell membrane.•Our work paved way for production of functional RXFP3 using E. coli overexpression. Relaxin family peptide receptor 3 (RXFP3) is an A-class G protein-coupled receptor. It is implicated in the regulation of food intake and stress response upon activation by the neuropeptide relaxin-3. So far, preparation of enough functional RXFP3 for structural analysis is still challenging due to its integral membrane protein nature. In the present study, we overexpressed an N-terminally secretory maltose-binding protein (sMBP)-fused shortened human RXFP3 in prokaryotic host Escherichia coli. A small fraction of the sMBP-RXFP3 fusion protein could be integrated into the E. coli cell membrane and this fraction retained ligand binding function although the measured binding parameters were different from those of sMBP-RXFP3 overexpressed in mammalian cells due to different cell membrane lipids of the different host cells. Our present work paved the way for production of functional RXFP3 using the low-cost E. coli expression system.