Purification and preparation of Marchantia polymorpha Auxin Response Factor 2 for phase separation studies

Eukaryotic cells compartmentalise to optimise their biochemistry. This occurs through membrane-surrounded compartments, but also through membraneless organelles (MLOs). Many MLOs arise from Liquid-Liquid Phase Separation (LLPS) of proteins. To investigate proteins for their capacity to phase separate, as well as what properties this results in, purified protein is required for in vitro phase separation assays. We have observed that Auxin Response Factor 2 from the common liverwort Marchantia polymorpha (MpARF2) forms nuclear assemblies in vivo. Auxin Response Factors (ARFs) are transcription factors crucial for the regulation of gene expression in response to auxin, an essential signalling molecule in plants. It is currently unclear what causes the formation of MpARF2 assemblies and how they affect the protein's function. To decipher this, we present the procedures for purifying MpARF2 and preparing it for in vitro studies. The observed MpARF2 assemblies could arise by phase separation. The properties of phase-separating proteins make their expression, purification and handling challenging; therefore, optimised procedures are required. Here, we show that the purification of soluble and monomeric full-length MpARF2, as well as (truncated) variants, is possible using a three-step purification procedure involving a protein solubility tag and buffers that suppress phase separation. We also describe the subsequent protein preparation that ensures reproducible phase separation of MpARF2 during in vitro experiments. These procedures pave the way for elucidating the phase separation properties of MpARF2 and the potential functional role of its assemblies while providing a protocol which can facilitate similar studies of other ARFs.