当前位置: X-MOL 学术Mol. Pharmaceutics › 论文详情
Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)
Charge Shielding Prevents Aggregation of Supercharged GFP Variants at High Protein Concentration
Molecular Pharmaceutics ( IF 4.9 ) Pub Date : 2017-09-18 00:00:00 , DOI: 10.1021/acs.molpharmaceut.7b00322
Joshua R. Laber 1 , Barton J. Dear 1 , Matheus L. Martins 1 , Devin E. Jackson 1 , Andrea DiVenere 1 , Jimmy D. Gollihar 1 , Andrew D. Ellington 1 , Thomas M. Truskett 1 , Keith P. Johnston 1 , Jennifer A. Maynard 1
Affiliation  

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.

中文翻译:

电荷屏蔽可防止在高蛋白浓度下聚集带电的GFP变体

了解蛋白质的稳定性对于抵抗蛋白质聚集疾病和开发新的蛋白质疗法至关重要。在生物系统中通常存在的高浓度下,纯化的蛋白质可能会表现出不良的高溶液粘度和不良的溶解性,这是由短程静电和疏水性蛋白质与蛋白质之间的相互作用所介导的。蛋白质氨基酸序列,蛋白质结构和溶剂条件之间的相互作用最小化了蛋白质与蛋白质之间的相互作用,这是设计行为良好的药用蛋白质的关键。但是,理论方法尚未产生解决这些问题的通用框架。在这里,我们分析了超级文件夹GFP(sfGFP)和两个增压的sfGFP变异体的高浓度行为,这些变异体的正式电荷为-18或+15。在低溶质条件下,sfGFP和-18变体形成了约10 mg / mL的凝胶或相分离物。在筛选表面电荷的条件下,包括具有高组氨酸或高NaCl浓度的制剂,所有三种变体在中等粘度下均可达到高达250 mg / mL的浓度。此外,在此范围内,所有三种变体均显示出非常相似的粘度-浓度曲线。高糖浓度不能模仿这种效果,而糖浓度会在不产生屏蔽电荷的情况下发挥排除体积的作用。总的来说,这些数据表明,电荷屏蔽不仅中和了长距离的静电相互作用,而且还由于表面电荷的各向异性而令人惊讶地中和了短距离的静电作用。这项工作表明,高离子强度下的sfGFP增压行为在很大程度上取决于粒子的几何形状,
更新日期:2017-09-19
down
wechat
bug