Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, USA.
Center for Synthetic Biology, Northwestern University, Evanston, IL, USA.
Nat Chem Biol. 2018 Jun;14(6):627-635. doi: 10.1038/s41589-018-0051-2. Epub 2018 May 7.
Glycosylation is an abundant post-translational modification that is important in disease and biotechnology. Current methods to understand and engineer glycosylation cannot sufficiently explore the vast experimental landscapes required to accurately predict and design glycosylation sites modified by glycosyltransferases. Here we describe a systematic platform for glycosylation sequence characterization and optimization by rapid expression and screening (GlycoSCORES), which combines cell-free protein synthesis and mass spectrometry of self-assembled monolayers. We produced six N- and O-linked polypeptide-modifying glycosyltransferases from bacteria and humans in vitro and rigorously determined their substrate specificities using 3,480 unique peptides and 13,903 unique reaction conditions. We then used GlycoSCORES to optimize and design small glycosylation sequence motifs that directed efficient N-linked glycosylation in vitro and in the Escherichia coli cytoplasm for three heterologous proteins, including the human immunoglobulin Fc domain. We find that GlycoSCORES is a broadly applicable method to facilitate fundamental understanding of glycosyltransferases and engineer synthetic glycoproteins.
糖基化是一种丰富的翻译后修饰，在疾病和生物技术中很重要。目前用于理解和设计糖基化的方法无法充分探索准确预测和设计糖基转移酶修饰的糖基化位点所需的广泛实验景观。在这里，我们描述了一种通过快速表达和筛选（GlycoSCORES）对糖基化序列进行表征和优化的系统平台，该平台结合了无细胞蛋白合成和自组装单层的质谱分析。我们在体外产生了六个来自细菌和人类的 N 和 O 连接多肽修饰糖基转移酶，并使用 3480 个独特肽和 13903 个独特反应条件严格确定了它们的底物特异性。然后，我们使用 GlycoSCORES 优化和设计了小的糖基化序列基序，这些基序在三种异源蛋白（包括人免疫球蛋白 Fc 结构域）的体外和大肠杆菌细胞质中指导有效的 N 连接糖基化。我们发现 GlycoSCORES 是一种广泛适用的方法，可以促进对糖基转移酶的基本理解和工程合成糖蛋白。
