The N‐terminal cleavage of fusion tags to restore the native N‐terminus of recombinant proteins is a challenging task and up to today, protocols need to be optimized for different proteins individually. Within this work, we present a novel protease that was designed in‐silico to yield enhanced promiscuity toward different N‐terminal amino acids. Two mutations in the active‐site amino acids of human Caspase‐2 were determined to increase the recognition of branched amino‐acids, which show only poor binding capabilities in the unmutated protease. These mutations were determined by sequential and structural comparisons of Caspase‐2 and Caspase‐3 and their effect was additionally predicted using free‐energy calculations. The two mutants proposed in the in‐silico studies were expressed and in‐vitro experiments confirmed the simulation results. Both mutants showed not only enhanced activities toward branched amino acids, but also smaller, unbranched amino acids. We believe that the created mutants constitute an important step toward generalized procedures to restore original N‐termini of recombinant fusion proteins.

中文翻译：

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通过合理设计增强 Caspase 蛋白酶家族成员的混杂性。


融合标签的N末端切割以恢复重组蛋白的天然N末端是一项具有挑战性的任务，到目前为止，需要针对不同的蛋白质单独优化方案。在这项工作中，我们提出了一种新型蛋白酶，该蛋白酶经过计算机设计，可增强对不同N末端氨基酸的混杂性。人 Caspase-2 活性位点氨基酸的两个突变被确定可以增加对支链氨基酸的识别，而在未突变的蛋白酶中，支链氨基酸的结合能力很差。这些突变是通过 Caspase-2 和 Caspase-3 的顺序和结构比较确定的，并且还使用自由能计算预测了它们的影响。计算机研究中提出的两种突变体得到表达，体外实验证实了模拟结果。两种突变体不仅表现出对支链氨基酸的增强活性，而且对较小的非支链氨基酸也表现出增强的活性。我们相信，所创建的突变体构成了恢复重组融合蛋白原始N末端的通用程序的重要一步。