Charge Shielding Prevents Aggregation of Supercharged GFP Variants at High Protein Concentration
| Autor: | Thomas M. Truskett, Jennifer A. Maynard, Matheus L. Martins, Joshua R. Laber, Jimmy Gollihar, Devin E. Jackson, Barton J. Dear, Andrea M. DiVenere, Keith P. Johnston, Andrew D. Ellington |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Protein Conformation Green Fluorescent Proteins Static Electricity Pharmaceutical Science Sequence (biology) Protein aggregation Protein Aggregation Pathological 030226 pharmacology & pharmacy Article Green fluorescent protein 03 medical and health sciences 0302 clinical medicine Protein structure Drug Discovery Surface charge Histidine Protein Stability Viscosity Chemistry Osmolar Concentration Solvent 030104 developmental biology Solubility Biochemistry Yield (chemistry) Biophysics Anisotropy Molecular Medicine Hydrophobic and Hydrophilic Interactions |
| Zdroj: | Molecular Pharmaceutics. 14:3269-3280 |
| ISSN: | 1543-8392 1543-8384 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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