UC Davis

As an RNA-binding protein, G3BP1 forms ribonuclear protein complexes with free mRNAs and proteins that leads to their separation from solution into biomolecular condensates. As a condensate, these species are removed from the processes of the cell thereby regulating transcription and metabolism which makes G3BP1 a compelling protein to study. In this paper, the foundation for producing isolated, in vitro G3BP1 is investigated for future spectroscopic and biochemical studies. Specifically, the human protein’s sequence was inserted into an E. coli plasmid designed for heterologous expression and immobilized metal affinity purification. Afterward, the affinity tag was cleaved, and the protein was isolated from other contaminants as verified by SDS-PAGE. Since the solution pH and concentrations of G3BP1 and sodium chloride conflicted with those of spectroscopic studies, additional tests were conducted to uncover the favorable conditions. Optimization of the expression, purification, and cleavage of the affinity tag of G3BP1 was successful in isolating and producing large enough quantities of the full-length and intact protein for further experiments but maintaining the viable protein in buffers amenable to solution state NMR proved challenging.