Charge Shielding Prevents Aggregation of Supercharged GFP Variants at High Protein Concentration

Abstract

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 solubility 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 design of 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 super-folder GFP (sfGFP) and two super-charged 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 concentrations, all three variants attained concentrations up to 250 mg/ml with moderate viscosities. Moreover, all three variants exhibited very similar viscosity-concentration profiles over this range. This effect was not mimicked by high sugar concentrations that exert excluded volume effects without shielding charge. Collectively, these data demonstrate that charge shielding not only neutralizes long-range electrostatic interactions but also, surprisingly, short-range electrostatic effects due to surface charge anisotropy. This work shows that supercharged sfGFP behavior under high ionic strength is largely determined by particle geometry, a conclusion that is supported by colloid models and may be applicable to pharmaceutically-relevant proteins.

Catalysis of Thermostable Alcohol Dehydrogenase Improved by Engineering the Microenvironment through Fusion with Supercharged Proteins

“…The reaction with −30 sfGFP–SpyTag was unsuccessful, 0 sfGFP–SpyTag went to apparent completion, and +36 sfGFP–SpyTag was partially reacted. To shield the effect of charge on the conjugations, the reactions were repeated in the presence of approximately 400 m m NaCl . The reactions went to apparent completion in all three cases, with SpyCatcher–AdhD being fully consumed resulting in the formation of sfGFP–SpyTag/SpyCatcher–AdhD with excess sfGFP–SpyTag (Figure S3 B).…”

Physicochemical Stability of Monoclonal Antibodies: A Review

“…The net effect of salts on protein stability is protein-dependent and is a complex balance of the multiple mechanisms by which the ionic salt interacts with protein molecules, shielding charged solvent exposed residues and then potentially decreasing protein-protein long-range electrostatic interactions. 47,81,122 This charge shielding may be responsible for a decrease in formulation viscosity, improving global colloidal stability. However, it may also favor short-range hydrophobic interactions at high protein concentration, reducing solubility.…”

“…However, it may also favor short-range hydrophobic interactions at high protein concentration, reducing solubility. 122 The effect of salts depends on their nature and concentration and may be divided between salting-in (stabilization) and salting-out (precipitation), high salt concentration (high ionic strength) being more in favor of aggregation salting-out. 123,124 The unchanged secondary structure analyses were however in favor of the retention of a native-like secondary structure even in the precipitated state, and mAb aggregation may be at least partially reversible by salt dilution.…”

Engineering high-yield biopolymer secretion creates an extracellular protein matrix for living materials

“…We note the apparent molecular weight of the purified SC (-) -ELP 60 -336c protein is approximately 10 kDa lower than seen in secreted protein prior to purification. We attribute this change in gel migration pattern to charge screening of these proteins by the PBS solution, which may decrease aggregation and/or alter protein conformation (56,57) . A higher molecular weight band associated with covalent bonding between SC (-) -ELP 60 -336c and RsaA 467 -ST in the control samples was not observed, which is consistent with the requirement for both SpyTag and SpyCatcher (-) to be present for ligation to occur.…”