Boivin Stephane, Kozak Sandra, Meijers Rob
European Molecular Biology Laboratory (EMBL), Hamburg Outstation, Notkestrasse 85, 22603 Hamburg, Germany.
Protein Expr Purif. 2013 Oct;91(2):192-206. doi: 10.1016/j.pep.2013.08.002. Epub 2013 Aug 12.
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.
重组蛋白的高效大规模生产依赖于在其分离纯化至均一性过程中对蛋白的精心处理。蛋白质稳定性低会因蛋白质降解、沉淀和折叠不稳定性导致纯化产率低。通常需要经过多次反复试验来优化蛋白质样品的均一性、稳定性和溶解性。我们已经建立了热荧光分析方法，以确定用于制备和表征单个蛋白质构建体的定制方案。我们采用两步法：首先筛选全局参数，然后寻找蛋白质特异性添加剂。第一次筛选的设计方式使得能够根据pH值、盐浓度、缓冲液类型和浓度辨别全局稳定性趋势。第二次筛选包含能够影响蛋白质构建体折叠、聚集状态和溶解性的小分子，还包括特异性结合并稳定蛋白质的小分子。这些筛选旨在评估纯化和储存方案，旨在为优化这些方案提供线索。自制的筛选方法已在欧洲分子生物学实验室汉堡分部的样品制备与表征（SPC）设施中对200多种不同的蛋白质构建体进行了测试。我们描述了哪些实时定量PCR仪可适用于进行热荧光分析，建立筛选所需的必要实验条件，一些解读数据的线索，并给出了热荧光分析在稳定性筛选之外的几个应用实例。
