Institute of Molecular Modeling and Simulation, University of Natural Resources and Life Sciences, Vienna, Austria.
Austrian Centre of Industrial Biotechnology, Vienna, Austria.
Proteins. 2020 Oct;88(10):1303-1318. doi: 10.1002/prot.25950. Epub 2020 Jun 11.
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.
融合标签的 N 端切割以恢复重组蛋白的天然 N 端是一项具有挑战性的任务，迄今为止，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，该蛋白酶是通过计算机设计的，旨在提高对不同 N 端氨基酸的广谱识别能力。通过对人 Caspase-2 的活性位点氨基酸进行两次突变，确定了增加对支链氨基酸的识别能力，在未突变的蛋白酶中，这些氨基酸的结合能力很差。这些突变是通过 Caspase-2 和 Caspase-3 的序列和结构比较确定的，并且使用自由能计算进一步预测了它们的效果。在计算机研究中提出的两个突变体被表达，并且体外实验证实了模拟结果。这两个突变体不仅对支链氨基酸表现出增强的活性，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，所创建的突变体是朝着恢复重组融合蛋白原始 N 端的通用程序迈出的重要一步。
