Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, Delaware, 19716.
Drug Product Development, Amgen Inc., Thousand Oaks, California, 91320.
Protein Sci. 2018 Jul;27(7):1275-1285. doi: 10.1002/pro.3415. Epub 2018 May 3.
Colloidal protein-protein interactions (PPI) are often expected to impact key behaviors of proteins in solution, such as aggregation rates and mechanisms, aggregate structure, protein solubility, and solution viscosity. PPI of an anti-fluorescein single chain antibody variable fragment (scFv) were characterized experimentally at low to intermediate ionic strength using a combination of static light scattering and sedimentation equilibrium ultracentrifugation. Surprisingly, the results indicated that interactions were strongly net-attractive and electrostatics promoted self-association. Only repulsive interactions were expected based on prior work and calculations based a homology model of a related scFv crystal structure. However, the crystal structure lacks the charged, net-neutral linker sequence. PyRosetta was used to generate a set of scFv structures with different linker conformations, and coarse-grained Monte Carlo simulations were used to evaluate the effect of different linker configurations via second osmotic virial coefficient (B ) simulations. The results show that the configuration of the linker has a significant effect on the calculated B values, and can result in strong electrostatic attractions between oppositely charged residues on the protein surface. This is particularly relevant for development of non-natural antibody products, where charged linkers and other loop regions may be prevalent. The results also provide a preliminary computational framework to evaluate the effect of unstructured linkers on experimental protein-protein interaction parameters such as B .
胶体蛋白-蛋白相互作用（PPI）通常被认为会影响蛋白质在溶液中的关键行为，如聚集速率和机制、聚集结构、蛋白质溶解度和溶液黏度。本研究在低至中等离子强度下，使用静态光散射和沉降平衡超速离心相结合的方法，实验表征了抗荧光素单链抗体可变片段（scFv）的 PPI。令人惊讶的是，结果表明相互作用具有强烈的净吸引力，静电作用促进了自组装。仅根据先前的工作和基于同源 scFv 晶体结构模型的计算，预计会存在排斥相互作用。然而，晶体结构缺乏带电的、净中性的连接序列。PyRosetta 用于生成一组具有不同连接子构象的 scFv 结构，粗粒化蒙特卡罗模拟用于通过第二渗透压第二维里系数（B ）模拟评估不同连接子构象的影响。结果表明，连接子的构象对计算出的 B 值有显著影响，并可能导致蛋白质表面上带相反电荷的残基之间产生强烈的静电吸引力。这对于开发非天然抗体产品尤为重要，因为在这些产品中，带电荷的连接子和其他环区可能很常见。该结果还提供了一个初步的计算框架，用于评估无规连接子对实验蛋白质-蛋白质相互作用参数（如 B ）的影响。
