The important roles that protein glycosylation plays in modulating the activities and efficacies of protein therapeutics have motivated the development of synthetic glycosylation systems in living bacteria and in vitro. A key challenge is the lack of glycosyltransferases that can efficiently and site-specifically glycosylate desired target proteins without the need to alter primary amino acid sequences at the acceptor site. Here, we report an efficient and systematic method to screen a library of glycosyltransferases capable of modifying comprehensive sets of acceptor peptide sequences in parallel. This approach is enabled by cell-free protein synthesis and mass spectrometry of self-assembled monolayers and is used to engineer a recently discovered prokaryotic N-glycosyltransferase (NGT). We screened 26 pools of site-saturated NGT libraries to identify relevant residues that determine polypeptide specificity and then characterized 122 NGT mutants, using 1052 unique peptides and 52,894 unique reaction conditions. We define a panel of 14 NGTs that can modify 93% of all sequences within the canonical X_–1-N-X₊₁-S/T eukaryotic glycosylation sequences as well as another panel for many noncanonical sequences (with 10 of 17 non-S/T amino acids at the X₊₂ position). We then successfully applied our panel of NGTs to increase the efficiency of glycosylation for three protein therapeutics. Our work promises to significantly expand the substrates amenable to in vitro and bacterial glycoengineering.

中文翻译：

---

使用高通量实验筛选具有改变特异性的 N-糖基转移酶

蛋白质糖基化在调节蛋白质治疗的活性和功效中发挥的重要作用推动了活细菌和体外合成糖基化系统的发展。一个关键的挑战是缺乏能够有效地、位点特异性地糖基化所需靶蛋白而不需要改变受体位点的一级氨基酸序列的糖基转移酶。在这里，我们报告了一种有效且系统的方法来筛选能够并行修饰全套受体肽序列的糖基转移酶文库。这种方法是通过无细胞蛋白质合成和自组装单层质谱分析实现的，并用于设计最近发现的原核N-糖基转移酶 (NGT)。我们筛选了 26 个位点饱和 NGT 文库，以鉴定决定多肽特异性的相关残基，然后使用 1052 个独特的肽和 52,894 个独特的反应条件表征了 122 个 NGT 突变体。我们定义了一个由 14 个 NGT 组成的面板，可以修改规范 X _–1 -NX ₊₁ -S/T 真核糖基化序列中所有序列的 93%，以及另一个用于许多非规范序列的面板（17 个非 S/ 中的 10 个） X ₊₂位置的T氨基酸）。然后，我们成功应用我们的 NGT 组来提高三种蛋白质疗法的糖基化效率。我们的工作有望显着扩展适合体外和细菌糖工程的底物。