Glycosylation is a critical post-translational protein modification that affects folding, half-life and functionality. Glycosylation is a non-templated and heterogeneous process because of the promiscuity of the enzymes involved. We describe a platform for sequential glycosylation reactions for tailored sugar structures (SUGAR-TARGET) that allows bespoke, controlled N-linked glycosylation in vitro enabled by immobilized enzymes produced with a one-step immobilization/purification method. We reconstruct a reaction cascade mimicking a glycosylation pathway where promiscuity naturally exists to humanize a range of proteins derived from different cellular systems, yielding near-homogeneous glycoforms. Immobilized β-1,4-galactosyltransferase is used to enhance the galactosylation profile of three IgGs, yielding 80.2–96.3% terminal galactosylation. Enzyme recycling is demonstrated for a reaction time greater than 80 h. The platform is easy to implement, modular and reusable and can therefore produce homogeneous glycan structures derived from various hosts for functional and clinical evaluation.

糖基化是一种重要的翻译后蛋白质修饰，影响折叠、半衰期和功能。由于所涉及的酶的混杂性，糖基化是一个非模板化的异质过程。我们描述了一种用于定制糖结构（SUGAR-TARGET）的连续糖基化反应的平台，该平台允许通过一步固定/纯化方法产生的固定化酶在体外进行定制的、受控的N-连接糖基化。我们重建了一个模仿糖基化途径的反应级联，其中自然存在混杂性，使来自不同细胞系统的一系列蛋白质人源化，产生近乎同质的糖形式。固定化 β-1,4-半乳糖基转移酶用于增强三种 IgG 的半乳糖基化特征，产生 80.2–96.3% 的末端半乳糖基化。酶回收被证明反应时间超过 80 小时。该平台易于实施、模块化且可重复使用，因此可以产生源自各种宿主的均质聚糖结构，用于功能和临床评估。