Abstract：
Cell-free protein synthesis (CFPS), whereby cell lysates are used to produce proteins from a genetic template, has matured as an attractive alternative to standard biomanufacturing modalities due to its high volumetric productivity contained within a distributable platform. Initially, cell-free lysates produced from Escherichia coli, which are both simple to produce and cost-effective for the production of a wide variety of proteins, were unable to produce glycosylated proteins as E. coli lacks native glycosylation machinery. With many important therapeutic proteins possessing asparagine-linked glycans that are critical for structure and function, this gap in CFPS production capabilities was addressed with the development of cell-free expression of glycoproteins (glycoCFE), which uses the supplementation of extracted lipid-linked oligosaccharides and purified oligosaccharyltransferases to enable glycoprotein production in the CFPS reaction environment. In this chapter, we highlight the basic methods for the preparation of reagents for glycoCFE and the protocol for expression and glycosylation of a model protein using a more productive, yet simplified, glycoCFE setup. Beyond this initial protocol, we also highlight how this protocol can be extended to a wide range of alternative glycan structures, oligosaccharyltransferases, and acceptor proteins as well as to a one-pot cell-free glycoprotein synthesis reaction.
摘要：
无细胞蛋白质合成（CFPS），细胞裂解物用于从遗传模板产生蛋白质，由于其可分配平台中包含的高体积生产率，因此已成为标准生物制造方式的有吸引力的替代方案。最初，从大肠杆菌产生的无细胞裂解物，既生产简单，又生产各种蛋白质具有成本效益，由于大肠杆菌缺乏天然糖基化机制,因此无法产生糖基化蛋白。许多重要的治疗性蛋白质具有天冬酰胺连接的聚糖，这些聚糖对结构和功能至关重要，CFPS生产能力的这种差距通过无细胞表达糖蛋白（glycoCFE）的发展来解决，其使用提取的脂质连接寡糖和纯化的寡糖转移酶的补充,以使得能够在CFPS反应环境中产生糖蛋白。在这一章中,我们重点介绍了制备glycoCFE试剂的基本方法，以及使用更高效的模型蛋白质表达和糖基化的方案，然而简化，gycoCFE设置。除了这个最初的协议，我们还强调了该协议如何扩展到广泛的替代聚糖结构，寡糖转移酶，和受体蛋白以及一锅法无细胞糖蛋白合成反应。
