Light-induced protein expression within droplet interface bilayers

尽管在膜蛋白的制备方面已经有了很大的进展，但这些蛋白通常很难处理，因为它们往往在水溶液中聚集。这些蛋白在靶膜附近的表达提供了另一种选择，因为将蛋白质掺入膜中可以防止其聚集。液滴界面双层(DIB)是一种有用的膜系统，因为它们提供了一个稳定的隔室，只封闭了很小的体积，并已被证明对快速筛选离子通道等应用是有用的。理想情况下，DIBs中的蛋白质表达将受到时间上的控制。在这项研究中，实现了一种光诱导的体外转录/翻译(IVTT)系统在液滴中。通过光触发表达，可以将IVTT系统传递到液滴，而不发生转录和存储准备按需表达膜蛋白的DIB。将应用两种策略：第一种策略利用表面活性剂azoTAB，它被证明在其反式构象中促进DNA缩合。光诱导的顺式异构化导致DNA的释放，从而允许转录。第二种策略旨在控制T7RNA聚合酶的活性，这是一种在各种IVTT系统中对转录至关重要的酶。在聚合酶的关键位置引入笼状氨基酸会导致失活的酶，这种酶可以不被激活，因此可以被不同波长的光激活。

Even though there has been substantial progress in the preparation of membrane proteins, these proteins are usually cumbersome to handle since they tend to aggregate in aqueous solution. Expression of these proteins in the vicinity of the target membrane provides an alternative as incorporation of the protein into the membrane can prevent its aggregation. Droplet interface bilayers (DIBs) are a useful membrane system since they provide a stable compartment, enclose only small volumes and have been shown to be useful for applications such as rapid screening of ion channels. Ideally, protein expression in DIBs would be temporally controlled. In this study, the implementation of a light-inducible in vitro transcription/translation (IVTT) system within droplets is pursued. Triggering the expression by light allows delivery of the IVTT system to droplets without transcription occurring and storage of DIBs ready to express membrane proteins on demand. Two strategies will be applied: The first one takes advantage of the surfactant azoTAB which was shown to facilitate DNA condensation in its trans conformation. Light-induced isomerization to the cis state leads to the release of DNA, permitting transcription. The second strategy aims to control the activity of T7 RNA polymerase, an enzyme crucial for transcription in various IVTT systems. Introducing a caged amino acid at a critical position in the polymerase will lead to an inactive enzyme which can be uncaged and therefore activated by light of a distinct wavelength.