近年来,已经出现了无细胞合成糖生物学技术,该技术能够在细胞范围外产生和重塑糖蛋白。然而,这些系统中的许多将多个合成步骤组合到一个锅中,其中可能存在竞争反应和副产物,这最终导致所需产物的低收率。在这项工作中,我们描述了一个集成无细胞蛋白质合成的微流控平台,糖基化,和在单独的隔室中纯化模型糖蛋白,其中每个步骤可以单独优化。微流体提供的优势,如反应区划分,可调的停留时间,束缚酶重复使用的能力,以及持续制造的潜力。此外,它为糖基化反应的时空控制提供了机会,这是用现有的基于细胞的和无细胞的糖基化系统难以实现的。在这项工作中,我们展示了一种基于流动的糖蛋白合成系统,该系统可促进无细胞蛋白质合成的增强,用固定化寡糖转移酶进行有效的蛋白质糖基化,和从无细胞裂解物中富集蛋白质产物。总的来说,这项工作代表了一种首创的糖基化芯片原型,可以用作蛋白质糖基化过程的机械解剖的实验室工具以及小批量的生物制造平台,分散式糖蛋白生产。
In recent years, cell-free synthetic glycobiology technologies have emerged that enable production and remodeling of glycoproteins outside the confines of the cell. However, many of these systems combine multiple synthesis steps into one pot where there can be competing reactions and side products that ultimately lead to low yield of the desired product. In this work, we describe a microfluidic platform that integrates cell-free protein synthesis, glycosylation, and purification of a model glycoprotein in separate compartments where each step can be individually optimized. Microfluidics offer advantages such as reaction compartmentalization, tunable residence time, the ability to tether enzymes for reuse, and the potential for continuous manufacturing. Moreover, it affords an opportunity for spatiotemporal control of glycosylation reactions that is difficult to achieve with existing cell-based and cell-free glycosylation systems. In this work, we demonstrate a flow-based glycoprotein synthesis system that promotes enhanced cell-free protein synthesis, efficient protein glycosylation with an immobilized
oligosaccharyltransferase, and enrichment of the protein product from cell-free lysate. Overall, this work represents a first-in-kind glycosylation-on-a-chip prototype that could find use as a laboratory tool for mechanistic dissection of the protein glycosylation process as well as a biomanufacturing platform for small batch, decentralized glycoprotein production.