Highlights•Optimization procedures

Highlights
•
Optimization procedures for sample purification and characterization.
•
Rational design of buffers and additive screens compatible with Thermofluor assays.
•
Interpretation of thermal shift assays and melting-temperature midpoint calculations.
Abstract
The efficient large scale production of recombinant proteins depends on the careful conditioning of the protein as it is isolated and purified to homogeneity. Low protein stability leads to low purification yields as a result of protein degradation, precipitation and folding instability. It is often necessary to go through several iterations of trial-and-error to optimize the homogeneity, stability and solubility of the protein sample. We have set up Thermofluor assays to identify customized protocols for the preparation and characterization of individual protein constructs. We apply a two-step approach: we first screen for global parameters, followed by a search for protein-specific additives. The first screen has been designed in such a way, that it is possible to discern global stability trends according to pH, salt concentration, buffer type and concentration. The second screen contains small molecules that can affect the folding, aggregation state and solubility of the protein construct and also includes small molecules that specifically bind and stabilize proteins. The screens are designed to evaluate purification and storage protocols, and aim to provide hints to optimize these protocols. The home-made screens have been tested on more than 200 different protein constructs at the Sample Preparation and Characterization (SPC) facility at EMBL Hamburg. We describe which RT-PCR machines can be adapted to perform Thermofluor assays, what are the necessary experimental conditions to set up a screen, some leads on how to interpret the data and we give several examples of Thermofluor applications beyond stability screens.

Abbreviations
ANS, Anilinonaphthalene-sulfonate; DSF, Differential Scanning Fluorimetry; DSC, Differential Scanning Calorimetry; TF, Thermofluor; RT-PCR, Reverse transcription polymerase chain reaction; Tm, Melting temperature
Keywords
Thermofluor; Differential scanning fluorimetry; Fluorescence thermal shift assay; Thermodenaturation; Protein stabilization; Buffer optimization; Additive screen; Thermostability; Melting point; Crystallization

結果 (中文) 1:

亮点•优化程序样品纯化与表征。•缓冲区和添加剂屏幕兼容 Thermofluor 检测方法的合理设计。•热位移检测和熔化温度中点计算的解释。摘要高效大规模生产重组蛋白取决于蛋白质小心空调作为它是分离和纯化。低蛋白质的稳定性，导致低纯化蛋白质的降解、 降水和折叠不稳定产量。通常，有的是需要经过反复的试验和错误进行优化的均匀性、 稳定性和溶解度的蛋白样品。我们有成立 Thermofluor 检测，识别为单个蛋白结构的制备及其自定义的协议。我们运用两步法： 我们第一个屏幕为全局参数，其后的检索，为特定的蛋白质添加剂。这样，就可以看出根据 ph 值、 盐浓度、 缓冲区类型和浓度的全球稳定趋势，设计了第一个屏幕。第二个屏幕包含可以影响折叠，聚集状态和溶解性的蛋白质结构的小分子，也包括具体绑定和稳定蛋白的小分子。屏幕旨在评估净化和存储协议，旨在提供提示，以优化这些协议。家庭自制的屏幕已经过测试的超过 200 种不同的蛋白质建于 EMBL 汉堡的样品制备和表征 (SPC) 设施。我们描述了哪些 RT-pcr 技术机器可以适应执行 Thermofluor 检测，有什么必要的实验条件，设置屏幕，一些关于如何解释数据潜在的顾客，我们给的 Thermofluor 应用程序稳定性屏幕之外的几个例子。缩写ANS，苯胺基萘 —-磺酸酯 ；DSF，差示扫描荧光分析法 ；DSC 差示扫描量热仪 ；TF，Thermofluor ；RT-pcr 技术，逆转录聚合酶链反应 ；Tm、 熔化温度关键字Thermofluor ；微分扫描荧光 ；荧光热位移检测 ；热变性 ；蛋白质稳定 ；缓冲区优化 ；添加剂的屏幕 ；热稳定性 ；熔点 ；结晶

結果 (中文) 3:

集锦
•
优化方法进行样品的纯化和表征
•
。设计合理的缓冲液和添加剂屏幕thermofluor检测兼容。
•
热转移分析解释和熔融温度计算
摘要
中点。高效的大规模生产重组蛋白质取决于蛋白质的精心调理是分离和纯化至均一。低蛋白的稳定性，导致较低的净化率作为一个结果，蛋白降解，沉淀和折叠的不稳定。通常需要通过试验和错误的几种迭代优化的均匀性，蛋白质样品的溶解度和稳定性。我们建立了分析鉴定thermofluor和个别蛋白质的制备表征构建定制的协议。我们采用两步法：全局参数，首先筛选，其次是一种特异的蛋白质添加剂搜索。第一个屏幕已被设计以这样一种方式，它是可以辨别的全局稳定性趋势根据pH，盐浓度，缓冲液的类型和浓度。第二屏幕包含会影响折叠的小分子，蛋白质的聚集状态和溶解性的构建还包括特异性结合和稳定蛋白质的小分子。屏幕设计进行净化和存储协议，旨在提供提示来优化这些协议。自制的屏幕已在超过200个不同的蛋白质测试结构在样品的制备和表征（SPC）在EMBL汉堡设备。我们描述了RT-PCR的机器可以实现thermofluor检测，必要的实验条件，建立一个屏幕是什么，一些线索，如何解释数据，我们给出几个应用实例thermofluor超越的稳定性屏幕。

缩写
ANS，anilinonaphthalene磺酸盐；DSF，差示扫描荧光法；DSC，差示扫描量热法；TF，thermofluor；RT-PCR，逆转录聚合酶链反应；TM，熔化温度
关键词
thermofluor；差示扫描荧光；荧光热转移法；热变性；蛋白质稳定；缓存优化；添加剂筛选；热稳定性；熔点；结晶