Light-induced protein expression within droplet interface bilayers

尽管在膜蛋白的制备方面已经有了实质性的进展，但这些蛋白通常难以处理，因为它们倾向于在水溶液中聚集。这些蛋白质在靶膜附近的表达提供了一种替代方案，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，因为它们提供稳定的隔室，仅封闭小体积，并且已经显示出可用于诸如快速筛选离子通道的应用。理想地，DIB中的蛋白质表达将被暂时控制。在这项研究中，实施的光诱导的体外转录/翻译（IVTT）系统内的液滴追求。通过光触发表达允许将IVTT系统递送至液滴而不发生转录，并储存准备好按需表达膜蛋白的DIB。将应用两种策略：第一种利用表面活性剂azoTAB，其显示出促进其反式构象中的DNA缩合。光诱导的异构化为顺式状态导致DNA的释放，允许转录。第二种策略旨在控制T7 RNA聚合酶的活性，T7 RNA聚合酶是一种在各种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将导致失活的酶，其可以被解除笼状，因此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.