Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.
Institute for Protein Design, University of Washington, Seattle, WA 98195, USA.
Science. 2020 Apr 3;368(6486):78-84. doi: 10.1126/science.aay2790.
The design of modular protein logic for regulating protein function at the posttranscriptional level is a challenge for synthetic biology. Here, we describe the design of two-input AND, OR, NAND, NOR, XNOR, and NOT gates built from de novo-designed proteins. These gates regulate the association of arbitrary protein units ranging from split enzymes to transcriptional machinery in vitro, in yeast and in primary human T cells, where they control the expression of the gene related to T cell exhaustion. Designed binding interaction cooperativity, confirmed by native mass spectrometry, makes the gates largely insensitive to stoichiometric imbalances in the inputs, and the modularity of the approach enables ready extension to three-input OR, AND, and disjunctive normal form gates. The modularity and cooperativity of the control elements, coupled with the ability to de novo design an essentially unlimited number of protein components, should enable the design of sophisticated posttranslational control logic over a wide range of biological functions.
在转录后水平调节蛋白质功能的模块化蛋白质逻辑设计是合成生物学面临的挑战。在这里，我们描述了由从头设计的蛋白质构建的两个输入 AND、OR、NAND、NOR、XNOR 和 NOT 门的设计。这些门在体外、酵母和原代人 T 细胞中调节任意蛋白质单元（包括分裂酶和转录机制）的结合，在这些细胞中，它们控制与 T 细胞耗竭相关的 基因的表达。通过天然质谱证实的设计结合相互作用的协同性使门对输入的化学计量不平衡具有很大的不敏感性，并且该方法的模块化可方便地扩展到三个输入 OR、AND 和析取范式门。控制元件的模块化和协同性，加上从头设计数量几乎不受限制的蛋白质组件的能力，应该能够在广泛的生物学功能范围内设计复杂的翻译后控制逻辑。
