Simultaneous positive and negative selection of proteases in bacterium based on cell suicide and antibiotic resistance
Simultaneous positive and negative selection of proteases in bacterium based on cell suicide and antibiotic resistance
复制标题
DOI：
10.1002/biot.202200488
复制
发表时间：
2023-03
影响因子：
4.7
通讯作者：
A. A. Yekeen-A.;Lu Zhang;Haiyan Liu;Quan Chen
中科院分区：
文献类型：
--
作者：
A. A. Yekeen-A.;Lu Zhang;Haiyan Liu;Quan Chen
用于工程化蛋白酶对治疗或工业相关的靶底物的特异性和选择性的方法受到广泛关注。在此，我们报告了一个细菌系统，用于同时检测和选择蛋白酶活性的阳性和阴性目标基板。该系统基于大肠杆菌中蛋白水解触发的细菌SuiCide和抗生素耐药性（PASCAR），利用β-内酰胺酶和人gasdermin D（GS）分别进行蛋白水解响应性阳性和阴性选择。正选择的适用性用烟草蚀纹病毒蛋白酶朝向非天然底物的识别的定向进化来说明。我们还利用了积极的选择计算重新设计的蛋白酶变体的有效评估。我们构建并优化了一系列GS突变体作为自杀模块-具有从高到低的选择严格性-这将使PASCAR能够用作实际适用的双重选择系统。这项研究提供了一个简单且易于使用的工具，有助于工程化的蛋白酶与定制的特异性和选择性。
Methods for engineering protease specificity and selectivity toward target substrates of therapeutic or industrial relevance are of wide interest. Herein, we report a bacterial system for the simultaneous detection and selection of protease activity on positive and negative target substrates. The system, based on Proteolysis‐triggered bActerial SuiCide and Antibiotic Resistance (PASCAR) in Escherichia coli, exploits the β‐lactamase and the human gasdermin D (GS) for the proteolysis‐responsive positive and negative selections, respectively. The applicability of the positive selection was illustrated with the directed evolution of the Tobacco etch virus protease toward the recognition of non‐native substrates. We also utilized the positive selection for the efficient evaluation of computationally redesigned protease variants. We constructed and optimized a series of GS mutants as suicide modules – with high to low selection stringencies – that would enable the use of PASCAR as a practically applicable dual selection system. This study provides a simple and easily accessible tool that facilitates the engineering of proteases with custom specificity and selectivity.