Latent Luciferase

 描述(申请人提供)：生物发光是一种强大、廉价和非侵入性的方法，可以在体外和体内监测基因表达、酶活性、蛋白质-蛋白质相互作用和对治疗药物的反应。然而，生物发光需要使用来自少量发光生物(例如萤火虫)的荧光素酶，并且是有限的 到基因操作既可能又实用的应用和生物体。在这项探索性/发展性拨款中，我们将确定非发光物种中的蛋白质是潜在的荧光素酶，当用适当的底物处理时，能够发出生物发光。为了支持这种底物导向的方法，我们已经鉴定了一种果蝇蛋白，当用合成的荧光素类似物处理时，它被发现是一种荧光素酶。这一史无前例的结果表明，蛋白质的功能可以通过简单地修改底物而不是突变来从根本上改变。除了对新的酶活性的进化和设计的范式转换的影响之外，非发光生物编码潜在的荧光素酶的前景对于内源过程的非侵入性光学成像具有变革性的潜力，潜在地使得在自然细胞和生物体中进行生物发光成像而不需要引入外源荧光素酶。

 DESCRIPTION (provided by applicant): Bioluminescence is a powerful, inexpensive, and non-invasive method to monitor gene expression, enzymatic activity, protein-protein interactions and response to therapeutics both in vitro and in vivo. However, bioluminescence requires the use of a luciferase derived from a small number of luminogenic organisms (e.g., fireflies), and is limited to applications and organisms where genetic manipulation is both possible and practical. In this exploratory/developmental grant, we will identify proteins in non-luminous species that are latent luciferases, capable of bioluminescent light emission when treated with an appropriate substrate. In support of this substrate-directed approach, we have identified a fruit fly protein that is revealed to be a luciferase when treated with a synthetic luciferin analog. This unprecedented result shows that the function of a protein can be fundamentally changed simply by modification of the substrate, rather than by mutation. Beyond the paradigm-shifting implications for the evolution and design of new enzymatic activities, the prospect that non-luminogenic organisms encode latent luciferases has transformative potential for the non-invasive optical imaging of endogenous processes, potentially enabling bioluminescence imaging in native cells and organisms without the need for the introduction of an exogenous luciferase.