High-throughput system for the thermostability analysis of proteins
用于蛋白质热稳定性分析的高通量系统
- 关键词：
- 来源：
- Protein Science
- 类型：
- 学术文献
- 语种：
- 英语
- 原文发布日期：
- 2024-05-27
- 摘要：
- Thermal stability of proteins is a primary metric for evaluating their physical properties. Although researchers attempted to predict it using machine learning frameworks, their performance has been dependent on the quality and quantity of published data. This is due to the technical limitation that thermodynamic characterization of protein denaturation by fluorescence or calorimetry in a high-throughput manner has been challenging. Obtaining a melting curve that derives solely from the target protein requires laborious purification, making it far from practical to prepare a hundred or more samples in a single workflow. Here, we aimed to overcome this throughput limitation by leveraging the high protein secretion efficacy of Brevibacillus and consecutive treatment with plate-scale purification methodologies. By handling the entire process of expression, purification, and analysis on a per-plate basis, we enabled the direct observation of protein denaturation in 384 samples within 4 days. To demonstrate a practical application of the system, we conducted a comprehensive analysis of 186 single mutants of a single-chain variable fragment of nivolumab, harvesting the melting temperature (Tm) ranging from −9.3 up to +10.8°C compared to the wild-type sequence. Our findings will allow for data-driven stabilization in protein design and streamlining the rational approaches.
- 所属专题：
- 173
