Post-translational control of genetic circuits using Potyvirus proteases.
Post-translational control of genetic circuits using Potyvirus proteases.
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DOI：
10.1093/nar/gkw537
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发表时间：
2016-07-27
影响因子：
14.9
通讯作者：
Voigt CA
中科院分区：
文献类型：
--
作者：
Fernandez-Rodriguez J;Voigt CA
基因工程项目通常需要控制蛋白质何时被降解。为此，我们使用了降解肽和失活肽之间的融合，这种融合可以添加到蛋白质的N端。当相应的蛋白酶表达时，它会裂解多肽，蛋白质就会被降解。马铃薯Y病毒的三种蛋白酶：裂解位点对在暴露退化、释放抑制结构域和裂解多聚蛋白方面具有正交性和活性。这个工具箱被应用于遗传电路的设计，作为控制调节器活性和降解的一种手段。首先，我们证明了一个门可以通过结构性表达一个失活的抑制子和有一个输入启动子驱动蛋白酶的表达来构建。研究还表明，抑制结构域的蛋白分解释放可以提高转录门的动态范围(200倍抑制)。接下来，我们设计了包含多个抑制子的多蛋白，并证明了它们的切割可以用来控制多个输出。最后，我们证明，通过添加一个被蛋白酶裂解的降解物，可以提高产量的动态范围(8倍到190倍)。因此，可控的蛋白质降解为调节和扩展合成基因电路的功能提供了一个强有力的工具。
Genetic engineering projects often require control over when a protein is degraded. To this end, we use a fusion between a degron and an inactivating peptide that can be added to the N-terminus of a protein. When the corresponding protease is expressed, it cleaves the peptide and the protein is degraded. Three protease:cleavage site pairs from Potyvirus are shown to be orthogonal and active in exposing degrons, releasing inhibitory domains and cleaving polyproteins. This toolbox is applied to the design of genetic circuits as a means to control regulator activity and degradation. First, we demonstrate that a gate can be constructed by constitutively expressing an inactivated repressor and having an input promoter drive the expression of the protease. It is also shown that the proteolytic release of an inhibitory domain can improve the dynamic range of a transcriptional gate (200-fold repression). Next, we design polyproteins containing multiple repressors and show that their cleavage can be used to control multiple outputs. Finally, we demonstrate that the dynamic range of an output can be improved (8-fold to 190-fold) with the addition of a protease-cleaved degron. Thus, controllable proteolysis offers a powerful tool for modulating and expanding the function of synthetic gene circuits.