Evolution of protease activation and specificity via alpha-2-macroglobulin-mediated covalent capture.
Evolution of protease activation and specificity via alpha-2-macroglobulin-mediated covalent capture.
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DOI：
10.1038/s41467-023-36099-7
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发表时间：
2023-02-11
影响因子：
16.6
通讯作者：
Hollfelder, Florian
中科院分区：
文献类型：
--
作者：
Knyphausen, Philipp;Rangel Pereira, Mariana;Brear, Paul;Hyvonen, Marko;Jermutus, Lutz;Hollfelder, Florian
调整蛋白酶的活性和特异性对于它们在工业、医学和研究中的应用至关重要。然而，工程或进化蛋白酶催化剂是具有挑战性的，往往是劳动密集型的。在此，我们描述了一种基于酵母显示的通用方法来加速这一过程。我们介绍了蛋白酶选择系统A2Mcap，该系统通过重新利用α -2-巨球蛋白（A2Ms）共价捕获蛋白酶催化剂。为了证明A2Mcap在蛋白酶工程中的应用，我们举例说明了六种丝氨酸蛋白酶的定向活性和特异性进化。这导致了金黄色葡萄球菌丝氨酸蛋白酶样（Spl）蛋白酶SplB的变异，SplB是一种用于重组蛋白加工的酶，不再需要通过去除n端信号肽来激活。基于模式的结构域重组用于绘制Spl蛋白酶的特异性决定区域，从而形成一个嵌合支架，支持通过子结构域交换进行特异性切换。A2Mcap能够克服定制蛋白酶过程中的关键挑战，这表明未来更容易获得此类试剂。定制蛋白酶可用于治疗、研究和生物技术应用。在这里，作者建立了一个基于细菌α -2巨球蛋白蛋白酶抑制剂的蛋白酶选择系统，并进化出葡萄球菌蛋白酶，以提高活性和改变特异性。
Tailoring of the activity and specificity of proteases is critical for their utility across industrial, medical and research purposes. However, engineering or evolving protease catalysts is challenging and often labour intensive. Here, we describe a generic method to accelerate this process based on yeast display. We introduce the protease selection system A2Mcap that covalently captures protease catalysts by repurposed alpha-2-macroglobulin (A2Ms). To demonstrate the utility of A2Mcap for protease engineering we exemplify the directed activity and specificity evolution of six serine proteases. This resulted in a variant of Staphylococcus aureus serin-protease-like (Spl) protease SplB, an enzyme used for recombinant protein processing, that no longer requires activation by N-terminal signal peptide removal. SCHEMA-based domain shuffling was used to map the specificity determining regions of Spl proteases, leading to a chimeric scaffold that supports specificity switching via subdomain exchange. The ability of A2Mcap to overcome key challenges en route to tailor-made proteases suggests easier access to such reagents in the future. Custom proteases find applications as therapeutics, in research and in biotechnological applications. Here, the authors establish a protease selection system based on bacterial alpha-2-macroglobulin protease inhibitors and evolve staphylococcal proteases for increased activity and altered specificity.