Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Tech E136, Evanston, IL, 60208, USA.
Center for Synthetic Biology, Northwestern University, 2145 Sheridan Road, Tech B486, Evanston, IL, 60208, USA.
Nat Commun. 2019 Nov 27;10(1):5404. doi: 10.1038/s41467-019-12024-9.
Glycosylation plays important roles in cellular function and endows protein therapeutics with beneficial properties. However, constructing biosynthetic pathways to study and engineer precise glycan structures on proteins remains a bottleneck. Here, we report a modular, versatile cell-free platform for glycosylation pathway assembly by rapid in vitro mixing and expression (GlycoPRIME). In GlycoPRIME, glycosylation pathways are assembled by mixing-and-matching cell-free synthesized glycosyltransferases that can elaborate a glucose primer installed onto protein targets by an N-glycosyltransferase. We demonstrate GlycoPRIME by constructing 37 putative protein glycosylation pathways, creating 23 unique glycan motifs, 18 of which have not yet been synthesized on proteins. We use selected pathways to synthesize a protein vaccine candidate with an α-galactose adjuvant motif in a one-pot cell-free system and human antibody constant regions with minimal sialic acid motifs in glycoengineered Escherichia coli. We anticipate that these methods and pathways will facilitate glycoscience and make possible new glycoengineering applications.
糖基化在细胞功能中起着重要作用，并赋予蛋白质治疗药物有益的特性。然而，构建用于研究和工程化蛋白质上精确糖基结构的生物合成途径仍然是一个瓶颈。在这里，我们报告了一种用于糖基化途径组装的模块化、多功能的无细胞平台，通过快速的体外混合和表达（GlycoPRIME）来实现。在 GlycoPRIME 中，糖基化途径是通过混合和匹配无细胞合成的糖基转移酶来组装的，这些酶可以在由 N-糖基转移酶安装在蛋白质靶标上的葡萄糖启动子上进行修饰。我们通过构建 37 种可能的蛋白质糖基化途径、创建 23 种独特的聚糖基序来证明 GlycoPRIME，其中 18 种基序尚未在蛋白质上合成。我们使用选定的途径在无细胞体系中一锅法合成了带有α-半乳糖佐剂基序的蛋白质疫苗候选物，以及在糖工程化大肠杆菌中带有最小唾液酸基序的人抗体恒定区。我们预计这些方法和途径将促进糖科学的发展，并为新的糖基工程应用提供可能。
