EXPRESSION AND PURIFICATION OF PLANT FIBRILLARIN FOR PHASE SEPARATION STUDIES

Fibrillarin is a key nucleolar protein involved in the synthesis and processing of ribosomal RNA (rRNA). It functions as an rRNA methyltransferase, facilitating the methylation of rRNA molecules, which is essential for the proper assembly and function of ribosomes. Fibrillarin also plays a role in the biogenesis of small nucleolar ribonucleoproteins (snoRNPs) and is important for maintaining nucleolar structure and function, thereby influencing overall plant growth and development. AtGRP2, a plant RNA-binding protein that plays a critical role in abiotic stress response, has been shown to localize to the nucleolus and potentially interact with AtFib1. We hypothesized that AtGRP2 may co-condense with AtFib1 within the nucleolus. To investigate this hypothesis, we expressed and purified three constructs of AtFib1.They included the full-length protein (residues 1-308), the N-terminal intrinsically disordered GAR domain (residues 1-68), and the C-terminal folded methyltransferase domain (residues 65-308). All constructs were cloned into the RP1B vector, which fuses the protein of interest with an N-terminal Thio₆His₆TEV expression/purification tag. AtFib1 constructs were expressed solubly in Escherichia coli BL21(DE3) at 18 °C for 18 hours. His₆-AtFib1_1-68, AtFib1_65-308, and AtFib1_1-308 were purified via nickel-affinity chromatography, followed by cleavage of the N-terminal tag using His₆-TEV protease. The cleaved His₆-tag and His₆-TEV protease were removed by a second nickel-affinity purification step, allowing the AtFib1 constructs to flow through the column. Finally, the AtFib1 constructs were further purified by size-exclusion chromatography. We are currently investigating the capacity of AtFib1 constructs to undergo phase separation in vitro and how this process is modulated by interaction with AtGRP2.