Skretas Georgios, Wood David W
Department of Chemical Engineering, Princeton University, Engineering Quadrangle, Olden St., Princeton, NJ 08544, USA.
Protein Sci. 2005 Feb;14(2):523-32. doi: 10.1110/ps.04996905. Epub 2005 Jan 4.
Inteins are the protein analogs of self-splicing RNA introns, as they post-translationally excise themselves from a variety of protein hosts. Intein insertion abolishes, in general, the activity of its host protein, which is subsequently restored upon intein excision. These protein elements therefore have the potential to be used as general molecular "switches" for the control of arbitrary target proteins. Based on rational design, an intein-based protein switch has been constructed whose splicing activity is conditionally triggered in vivo by the presence of thyroid hormone or synthetic analogs. This modified intein was used in Escherichia coli to demonstrate that a number of different proteins can be inactivated by intein insertion and then reactivated by the addition of thyroid hormone via ligand-induced splicing. This conditional activation was also found to occur in a dose-dependent manner. Rational protein engineering was then combined with genetic selection to evolve an additional intein whose activity is controlled by the presence of synthetic estrogen ligands. The ability to regulate protein function post-translationally through the use of ligand-controlled intein splicing will most likely find applications in metabolic engineering, drug discovery and delivery, biosensing, molecular computation, as well as many additional areas of biotechnology.
内含肽是自我剪接RNA内含子的蛋白质类似物，因为它们在翻译后从多种蛋白质宿主中自我切除。一般来说，内含肽的插入会消除其宿主蛋白质的活性，而这种活性会在内含肽切除后恢复。因此，这些蛋白质元件有潜力用作控制任意靶蛋白的通用分子“开关”。基于合理设计，构建了一种基于内含肽的蛋白质开关，其剪接活性在体内由甲状腺激素或合成类似物的存在有条件地触发。这种修饰的内含肽在大肠杆菌中使用，以证明许多不同的蛋白质可以通过内含肽插入而失活，然后通过添加甲状腺激素经配体诱导剪接而重新激活。这种条件激活也呈剂量依赖性。然后将合理的蛋白质工程与基因筛选相结合，进化出另一种内含肽，其活性由合成雌激素配体的存在控制。通过使用配体控制的内含肽剪接在翻译后调节蛋白质功能的能力很可能在代谢工程、药物发现与递送、生物传感、分子计算以及许多其他生物技术领域找到应用。
