State Key Laboratory of Surface Physics, Multiscale Research Institute of Complex Systems, Department of Physics, Fudan University, Shanghai 200433, China.
School of Life Science, Fudan University, Shanghai 200433, China.
Int J Mol Sci. 2021 Mar 9;22(5):2768. doi: 10.3390/ijms22052768.
Protein engineering is actively pursued in industrial and laboratory settings for high thermostability. Among the many protein engineering methods, rational design by bioinformatics provides theoretical guidance without time-consuming experimental screenings. However, most rational design methods either rely on protein tertiary structure information or have limited accuracies. We proposed a primary-sequence-based algorithm for increasing the heat resistance of a protein while maintaining its functions. Using adenylate kinase (ADK) family as a model system, this method identified a series of amino acid sites closely related to thermostability. Single- and double-point mutants constructed based on this method increase the thermal denaturation temperature of the mesophilic () ADK by 5.5 and 8.3 °C, respectively, while preserving most of the catalytic function at ambient temperatures. Additionally, the constructed mutants have improved enzymatic activity at higher temperature.
蛋白质工程在工业和实验室环境中被积极追求以获得高耐热性。在许多蛋白质工程方法中，生物信息学的合理设计提供了理论指导，而无需耗时的实验筛选。然而，大多数合理设计方法要么依赖于蛋白质三级结构信息，要么准确性有限。我们提出了一种基于一级序列的算法，用于在保持蛋白质功能的同时提高其耐热性。使用腺嘌呤激酶（ADK）家族作为模型系统，该方法确定了一系列与耐热性密切相关的氨基酸位点。基于该方法构建的单点和双点突变体分别将中温（）ADK 的热变性温度提高了 5.5 和 8.3°C，同时在环境温度下保留了大部分催化功能。此外，构建的突变体在较高温度下具有提高的酶活性。
