Cell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for accelerating the design of cellular function, on-demand biomanufacturing, portable diagnostics, and educational kits. Many essential biological processes that could endow CFE systems with desired functions, such as protein glycosylation, rely on the activity of membrane-bound components. However, without the use of synthetic membrane mimics, activating membrane-dependent functionality in bacterial CFE systems remains largely unstudied. Here, we address this gap by characterizing native, cell-derived membrane vesicles in Escherichia coli-based CFE extracts and describing methods to enrich vesicles with heterologous, membrane-bound machinery. We first use nanocharacterization techniques to show that lipid vesicles in CFE extracts are tens to hundreds of nanometers across, and on the order of ~3x10¹² particles/mL. We then determine how extract processing methods, such as post-lysis centrifugation, can be used to modulate concentrations of membrane vesicles in CFE systems. By tuning these methods, we show that increasing the number of vesicle particles to ~7x10¹² particles/mL can be used to increase concentrations of heterologous membrane protein cargo expressed prior to lysis. Finally, we apply our methods to enrich membrane-bound oligosaccharyltransferases and lipid-linked oligosaccharides for improving N-linked and O-linked glycoprotein synthesis. We anticipate that our findings will facilitate in vitro gene expression systems that require membrane-dependent activities and open new opportunities in glycoengineering.

中文翻译：

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通过表征和富集天然膜囊泡来改善无细胞糖蛋白的合成

粗细胞提取物的无细胞基因表达（CFE）系统已被广泛关注，以加速细胞功能设计，按需生物制造，便携式诊断和教育工具包。可以使CFE系统具有所需功能（例如蛋白质糖基化）的许多基本生物学过程都依赖于膜结合成分的活性。但是，在不使用合成膜模拟物的情况下，细菌CFE系统中依赖于膜的活化功能仍未得到充分研究。在这里，我们通过表征大肠杆菌中天然的，细胞衍生的膜囊泡来解决这一差距的CFE提取物，并描述了利用异源的膜结合机器富集囊泡的方法。我们首先使用纳米表征技术来显示CFE提取物中的脂质囊泡跨度为数十至数百纳米，大约为3x10 ¹²颗粒/ mL。然后，我们确定如何使用提取物处理方法（如裂解后离心）来调节CFE系统中膜囊泡的浓度。通过调整这些方法，我们表明将囊泡颗粒的数量增加至〜7x10 ¹²颗粒/ mL可用于增加裂解前表达的异源膜蛋白货物的浓度。最后，我们运用我们的方法来富集膜结合的寡糖基转移酶和脂质连接的寡糖，以改善N链和O链糖蛋白合成。我们预期我们的发现将促进需要膜依赖活性的体外基因表达系统，并在糖工程中打开新的机会。