Protein glycosylation, the enzymatic modification of amino acid sidechains with sugar moieties, plays critical roles in cellular function, human health, and biotechnology. However, studying and producing defined glycoproteins remains challenging. Cell-free glycoprotein synthesis systems, in which protein synthesis and glycosylation are performed in crude cell extracts, offer new approaches to address these challenges. Here, we review versatile, state-of-the-art systems for biomanufacturing glycoproteins in prokaryotic and eukaryotic cell-free systems with natural and synthetic N-linked glycosylation pathways. We discuss existing challenges and future opportunities in the use of cell-free systems for the design, manufacture, and study of glycoprotein biomedicines.

蛋白质糖基化是用糖基对氨基酸侧链进行酶促修饰，在细胞功能、人类健康和生物技术中发挥着关键作用。然而，研究和生产确定的糖蛋白仍然具有挑战性。无细胞糖蛋白合成系统，其中蛋白质合成和糖基化在粗细胞提取物中进行，提供了应对这些挑战的新方法。在这里，我们回顾了用于在原核和真核无细胞系统中通过天然和合成的N连接糖基化途径生物制造糖蛋白的多功能、最先进的系统。我们讨论了使用无细胞系统设计、制造和研究糖蛋白生物药物的现有挑战和未来机遇。