Department of Biology, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, University of São Paulo, São Paulo, SP, Brazil.
Department of Microbial Biochemistry and Genomics, Biological Research Institute Clemente Estable, Montevideo, Uruguay.
Biomed Res Int. 2019 Jan 3;2019:4798793. doi: 10.1155/2019/4798793. eCollection 2019.
All biosensing platforms rest on two pillars: specific biochemical recognition of a particular analyte and transduction of that recognition into a readily detectable signal. Most existing biosensing technologies utilize proteins that passively bind to their analytes and therefore require wasteful washing steps, specialized reagents, and expensive instruments for detection. To overcome these limitations, protein engineering strategies have been applied to develop new classes of protein-based sensor/actuators, known as protein switches, responding to small molecules. Protein switches change their active state (output) in response to a binding event or physical signal (input) and therefore show a tremendous potential to work as a biosensor. Synthetic protein switches can be created by the fusion between two genes, one coding for a sensor protein (input domain) and the other coding for an actuator protein (output domain) by domain insertion. The binding of a signal molecule to the engineered protein will switch the protein function from an "off" to an "on" state (or vice versa) as desired. The molecular switch could, for example, sense the presence of a metabolite, pollutant, or a biomarker and trigger a cellular response. The potential sensing and response capabilities are enormous; however, the recognition repertoire of natural switches is limited. Thereby, bioengineers have been struggling to expand the toolkit of molecular switches recognition repertoire utilizing periplasmic binding proteins (PBPs) as protein-sensing components. PBPs are a superfamily of bacterial proteins that provide interesting features to engineer biosensors, for instance, immense ligand-binding diversity and high affinity, and undergo large conformational changes in response to ligand binding. The development of these protein switches has yielded insights into the design of protein-based biosensors, particularly in the area of allosteric domain fusions. Here, recent protein engineering approaches for expanding the versatility of protein switches are reviewed, with an emphasis on studies that used PBPs to generate novel switches through protein domain insertion.
对特定分析物的特定生化识别和将这种识别转化为易于检测的信号。大多数现有的生物传感技术利用被动结合其分析物的蛋白质，因此需要浪费的洗涤步骤、专用试剂和昂贵的仪器进行检测。为了克服这些限制，已经应用蛋白质工程策略来开发新的蛋白质基传感器/执行器类别，称为蛋白质开关，以响应小分子。蛋白质开关响应结合事件或物理信号（输入）改变其活性状态（输出），因此具有作为生物传感器工作的巨大潜力。合成蛋白质开关可以通过两个基因的融合来创建，一个基因编码传感器蛋白（输入域），另一个基因编码执行器蛋白（输出域），通过插入域。信号分子与工程化蛋白质的结合将使蛋白质功能从“关闭”状态切换到“开启”状态（或反之亦然），如所需。分子开关可以例如感测代谢物、污染物或生物标志物的存在并触发细胞反应。潜在的传感和响应能力是巨大的；然而，天然开关的识别范围有限。因此，生物工程师一直在努力利用周质结合蛋白 (PBP) 作为蛋白质传感组件来扩展分子开关的识别范围工具包。PBP 是细菌蛋白的超家族，为工程生物传感器提供了有趣的特征，例如巨大的配体结合多样性和高亲和力，以及在配体结合时发生大的构象变化。这些蛋白质开关的开发为基于蛋白质的生物传感器的设计提供了深入的了解，特别是在变构域融合领域。在这里，回顾了用于扩展蛋白质开关多功能性的最新蛋白质工程方法，重点介绍了使用 PBP 通过蛋白质域插入生成新型开关的研究。
