Eukaryotic cell-free protein synthesis (CFPS) systems have the potential to simplify and speed up the expression and high-throughput analysis of complex proteins with functionally relevant post-translational modifications (PTMs). However, low yields and the inability to scale such systems have so far prevented their widespread adoption in protein research and manufacturing. Here, we present a detailed demonstration for the capabilities of a CFPS system derived from Nicotiana tabacum BY-2 cell culture (BY-2 lysate; BYL). BYL is able to express diverse, functional proteins at high yields in under 48 hours, complete with native disulfide bonds and N-glycosylation. An optimised version of the technology is commercialised as 'ALiCE', engineered for high yields of up to 3 mg/mL. Recent advances in the scaling of BYL production methodologies have allowed scaling of the CFPS reaction. We show simple, linear scale-up of batch mode reporter proten expression from a 100 uL microtiter plate format to 10 mL and 100 mL volumes in standard Erlenmeyer flasks, culminating in preliminary data from 1 L reactions in a CELL-tainer CT20 rocking motion bioreactor. As such, these works represent the first published example of a eukaryotic CFPS reaction scaled past the 10 mL level by several orders of magnitude. We show the ability of BYL to produce the simple reporter protein eYFP and large, multimeric virus-like particles directly in the cytosolic fraction. Complex proteins are processed using the native microsomes of BYL and functional expression of multiple classes of complex, difficult-to-express proteins is demonstrated, specifically: a dimeric, glycoprotein enzyme, glucose oxidase; the monoclonal antibody adalimumab; the SARS-Cov-2 receptor-binding domain; human epidermal growth factor; and a G protein-coupled receptor membrane protein, cannabinoid receptor type 2. Functional binding and activity are shown using a combination of surface plasmon resonance techniques, a serology-based ELISA method and a G protein activation assay. Finally, in-depth post-translational modification (PTM) characterisation of purified proteins through disulfide bond and N-glycan analysis is also revealed - previously difficult in the eukaryotic CFPS space due to limitations in reaction volumes and yields. Taken together, BYL provides a real opportunity for screening of complex proteins at the microscale with subsequent amplification to manufacturing-ready levels using off-the-shelf protocols. This end-to-end platform suggests the potential to significantly reduce cost and the time-to-market for high value proteins and biologics.

中文翻译：

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ALiCE：多功能、高产和可扩展的真核无细胞蛋白质合成 (CFPS) 系统

真核无细胞蛋白质合成 (CFPS) 系统具有简化和加速具有功能相关翻译后修饰 (PTM) 的复杂蛋白质的表达和高通量分析的潜力。然而，迄今为止，低产量和无法扩大此类系统的规模阻碍了它们在蛋白质研究和制造中的广泛应用。在这里，我们详细演示了源自烟草 BY-2 细胞培养物（BY-2 裂解物；BYL）的 CFPS 系统的功能。BYL 能够在 48 小时内以高产量表达多种功能性蛋白质，并完成天然二硫键和 N-糖基化。该技术的优化版本被商业化为“ALiCE”，专为高达 3 mg/mL 的高产量而设计。BYL 生产方法规模化的最新进展允许 CFPS 反应规模化。我们展示了在标准锥形瓶中从 100 uL 微量滴定板格式到 10 mL 和 100 mL 体积的批处理模式报告蛋白表达的简单线性放大，最终在 CELL-tainer CT20 摇摆运动生物反应器中获得 1 L 反应的初步数据. 因此，这些作品代表了第一个发表的真核 CFPS 反应的例子，其规模超过 10 mL 水平几个数量级。我们展示了 BYL 直接在细胞溶质部分中产生简单报告蛋白 eYFP 和大型多聚体病毒样颗粒的能力。使用 BYL 的天然微粒体加工复杂蛋白质，并证明了多类复杂、难以表达的蛋白质的功能表达，特别是：二聚体、糖蛋白酶、葡萄糖氧化酶；单克隆抗体阿达木单抗；SARS-Cov-2受体结合域；人表皮生长因子；和 G 蛋白偶联受体膜蛋白，即 2 型大麻素受体。结合使用表面等离子共振技术、基于血清学的 ELISA 方法和 G 蛋白活化测定显示功能结合和活性。最后，还揭示了通过二硫键和 N-聚糖分析对纯化蛋白质进行深入的翻译后修饰 (PTM) 表征 - 由于反应体积和产量的限制，以前在真核 CFPS 空间中是困难的。总之，BYL 提供了一个真正的机会，可以在微尺度上筛选复杂的蛋白质，然后使用现成的协议将其放大到可制造的水平。