Charge Shielding Prevents Aggregation of Supercharged GFP Variants at High Protein Concentration.
Charge Shielding Prevents Aggregation of Supercharged GFP Variants at High Protein Concentration.
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DOI：
10.1021/acs.molpharmaceut.7b00322
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发表时间：
2017-10-02
影响因子：
4.9
通讯作者：
Maynard JA
中科院分区：
文献类型：
--
作者：
Laber JR;Dear BJ;Martins ML;Jackson DE;DiVenere A;Gollihar JD;Ellington AD;Truskett TM;Johnston KP;Maynard JA
了解蛋白质稳定性对于抗击蛋白质聚集性疾病和开发新的蛋白质疗法至关重要。在生物体系中经常存在的高浓度情况下，纯化的蛋白质可能会表现出不希望看到的高溶液粘度和较差的溶解性，这是由短程静电和疏水蛋白质-蛋白质相互作用所介导的。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以减少蛋白质与蛋白质之间的相互作用是设计性能良好的药物蛋白质的关键。然而，理论方法尚未产生解决这些问题的一般框架。在这里，我们分析了超折叠绿色荧光蛋白(SfGFP)和两个形式电荷为−18或+15的超级带电的sfGFP变体的高浓度行为。在低共溶液条件下，sfGFP和−18变体形成的凝胶或相分离于~10 mg/ml。在筛选表面电荷的条件下，包括高组氨酸或高NaC l浓度的配方，所有这三种变体都达到了浓度高达250 mg/ml的中等粘度。此外，在这一范围内，所有三种变种都表现出非常相似的粘度-浓度曲线。这种效应不能被没有屏蔽电荷而产生排除体积效应的高糖浓度所模拟。总而言之，这些数据表明，电荷屏蔽不仅中和了长程静电相互作用，而且令人惊讶的是，由于表面电荷各向异性，还产生了短程静电效应。这项工作表明，高离子强度下sfGFP的超电荷行为在很大程度上取决于粒子的几何形状，这一结论得到了胶体模型的支持，并可能适用于与药物相关的蛋白质。
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.
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影响因子：
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作者：
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通讯作者：
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