1 research outputs found
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 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 neutralizes not only
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