Investigation of the thermal shift assay and its power to predict protein and virus stabilizing conditions
Investigation of the thermal shift assay and its power to predict protein and virus stabilizing conditions
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DOI：
10.1016/j.jpba.2018.08.017
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发表时间：
2018-11-30
影响因子：
3.4
通讯作者：
Brgles, Marija
中科院分区：
文献类型：
--
作者：
Sviben, Dora;Bertosa, Branimir;Brgles, Marija
蛋白质热漂移分析(TSA)已被广泛应用于蛋白质稳定性的研究(蛋白质生物药物稳定、蛋白质结晶研究或重组蛋白质的筛选)和药物发现(筛选配体或抑制剂)。这项工作旨在分析热位移分析结果与蛋白质聚合(多聚体和聚集)倾向的比较，并测试最稳定的配方对包膜病毒的稳定作用。考察了蛋白质浓度、缓冲液pH值和摩尔比对三种蛋白质(免疫球蛋白G、卵清蛋白和白蛋白)的影响，结果表明这些因素都对蛋白质熔点T-m的测定有影响，对这三种蛋白质的影响是相似的。以卵清蛋白为例，进行了分子动力学模拟，目的是了解蛋白质热稳定性依赖于pH的分子基础。三种变性剂在较宽的浓度范围内对T-m的影响很好地表明，只有在最高浓度时才发生化学变性。结果表明，大多数制剂对不同蛋白质的T-m影响相似。使用细胞培养感染力试验(CCID50)对大多数成功的制剂进行了包膜病毒稳定潜力的测试，结果显示与TSA结果缺乏相关性。只有通过SEC-HPLC法测量的T-m位移与蛋白质聚合的弱相关性，这意味着不能从T-m位移来预测聚合。(C)2018爱思唯尔B.V.保留所有权利。
Protein thermal shift assay (TSA) has been extensively used in investigation of protein stabilization (for protein biopharmaceutics stabilization, protein crystallization studies or screening of recombinant proteins) and drug discovery (screening of ligands or inhibitors). This work aimed to analyze thermal shift assay results in comparison to protein polymerization (multimerization and aggregation) propensity and test the most stabilizing formulations for their stabilization effect on enveloped viruses. Influence of protein concentration, buffer pH and molarity was tested on three proteins (immunoglobulin G, ovalbumin, and albumin) and results showed that each of these factors has an impact on determined shift in protein melting point T-m, and the impact was similar for all three proteins. In case of ovalbumin, molecular dynamics simulations were performed with the goal to understanding molecular basis of protein's thermal stability dependence on pH. Effect of three denaturing agents in a wide concentration range on T-m, showed nicely that chemical denaturation occurs only at the highest concentrations. Results showed similar effect on T-m for most formulations on different proteins. Most successful formulations were tested for enveloped virus stabilizing potential using cell culture infectivity assay (CCID50) and results showed lack of correlation with TSA results. Only weak correlation of T-m shift and protein polymerization measured by SEC-HPLC was obtained, meaning that polymerization cannot be predicted from T. shifts. (C) 2018 Elsevier B.V. All rights reserved.