Relaxin is a peptide hormone that has demonstrated positive effects in the treatment of acute heart failure, fibrosis, and scleroderma. Relaxin is the endogenous agonist to RXFP1, an atypical class A G protein-coupled receptor (GPCR), possessing a unique extracellular domain (ECD) and mechanism of activation, which involves a series of complex conformational changes within the ECD. This mechanism is not well understood due to the lack of an atomic-resolution structure of RXFP1. Structural studies of RXFP1, and many GPCRs is difficult because of low receptor expression in recombinant systems making it difficult to purify for structural studies. Structures of RXFP1 would allow us to understand this unique mechanism of activation and facilitate rational drug design targeting RXFP1.
We developed a high-throughput mammalian directed evolution platform to introduce and select for mutations that resulted in increased GPCR expression. Thus, the aim is to use this platform to generate high expressing mutants of RXFP1 to facilitate cryo-EM studies.
To purify RXFP1, an Expi293 cell line stably expressing RXFP1 was generated and cells were harvested prior to purification. The cell pellets were lysed, solubilized in detergent, and purified using FLAG affinity purification and size exclusion chromatography. While final purity was excellent, yields were insufficient for cryo-EM studies.
Using our directed evolution platform, we generated a mutant RXFP1 library containing over 350,000 clones and used fluorescence activated cell sorting in the presence of fluorescently labelled relaxin to isolate and enrich the high expressing population. Isolated mutants demonstrated a 50 % increase in recombinant expression compared to WT. These mutants retained their ability to bind and signal like WT and contained 3-5 mutations per clone. Moving forward, clones will be subject to a second round of evolution to further increase recombinant expression. The best mutant will be used for downstream purification and cryo-EM studies.