Charge shielding prevents aggregation of supercharged GFP variants at high protein concentration

Understanding protein stability is central to combatting protein aggregation diseases and developing new protein therapeutics. At the high concentrations often present in biological systems, purified proteins can exhibit undesirable high solution viscosities and poor solubilities mediated by short-range electrostatic and hydrophobic protein–protein interactions. The interplay between protein amino acid sequence, protein structure, and solvent conditions to minimize protein–protein interactions is key to designing well-behaved pharmaceutical proteins. However, theoretical approaches have yet to yield a general framework to address these problems. Here, we analyzed the high concentration behavior of superfolder GFP (sfGFP) and two supercharged sfGFP variants engineered to have formal charges of −18 or +15. Under low cosolute conditions, sfGFP and the −18 variant formed a gel or phase separated at ∼10 mg/mL. Under conditions that screen surface charges, including formulations with high histidine or high NaCl...