GST-FUS PURIFICATION STRATEGY FOR BIOPHYSICAL STUDIES

Fused in Sarcoma (FUS) is an RNA-binding protein involved in RNA splicing, transcription regulation, and DNA repair. FUS dysfunction is related to several neurodegenerative diseases, including amyotrophic lateral sclerosis. Thus, in vitro biophysical studies are crucial for understanding FUS (mis-)function under different physicochemical conditions. However, preparing recombinant full-length FUS for in vitro studies is quite challenging and requires additional care because its extensive intrinsically disordered regions (IDRs) are prone to proteolytic degradation and aggregation. Although N-terminal GST-tagged FUS (GST-FUS) constructs are widely used, detailed information regarding purification efficiency, protein yield, and sample integrity is rarely reported or remains insufficiently described. Here, we sought to optimize a reproducible expression and purification pipeline for full-length FUS and to identify the major limitations associated with the conventional GST-FUS strategy. GST-FUS expression was screened in different E. coli strains across a range of IPTG concentrations and induction temperatures. Expression was successfully achieved in E. coli T7 Express(DE3), as confirmed by analysis on Coomassie-stained SDS-PAGE. Cell-lysis was performed under different physicochemical conditions, including low and high ionic strengths, the presence or absence of Triton X-100, and phosphate-buffered saline. Under both low- and high-salt conditions, a substantial fraction of the protein partitioned into inclusion bodies. In PBS, soluble GST-FUS could be recovered by glutathione affinity chromatography and eluted with 20 mM reduced glutathione. Because extensive proteolysis was observed throughout the purification workflow, we tested a second construct containing an N-terminal GST tag and C-terminal GFP/Histag as a strategy to selectively recover intact full-length FUS and exclude proteolytic fragments. For this construct, a small-scale screening of purification conditions was performed using a 24-well format varying pH and ionic strength. Size-exclusion chromatography is being optimized to obtain a homogeneous sample. After obtaining purified full-length FUS samples, we aim to perform biophysical characterization experiments, including dynamic light scattering and differential scanning calorimetry, to examine their structural and functional properties, and a phase separation assay to understand the behavior of this protein under different conditions.