Robert F. Smith School of Chemical and Biomolecular Engineering, Cornell University, 120 Olin Hall, Ithaca, NY, 14853, USA.
Cornell Institute of Biotechnology, Cornell University, Ithaca, NY, 14853, USA.
Nat Commun. 2022 Oct 24;13(1):6325. doi: 10.1038/s41467-022-34029-7.
The ability to reconstitute natural glycosylation pathways or prototype entirely new ones from scratch is hampered by the limited availability of functional glycoenzymes, many of which are membrane proteins that fail to express in heterologous hosts. Here, we describe a strategy for topologically converting membrane-bound glycosyltransferases (GTs) into water soluble biocatalysts, which are expressed at high levels in the cytoplasm of living cells with retention of biological activity. We demonstrate the universality of the approach through facile production of 98 difficult-to-express GTs, predominantly of human origin, across several commonly used expression platforms. Using a subset of these water-soluble enzymes, we perform structural remodeling of both free and protein-linked glycans including those found on the monoclonal antibody therapeutic trastuzumab. Overall, our strategy for rationally redesigning GTs provides an effective and versatile biosynthetic route to large quantities of diverse, enzymatically active GTs, which should find use in structure-function studies as well as in biochemical and biomedical applications involving complex glycomolecules.
从头重建天然糖基化途径或原型的能力受到功能糖基酶可用性有限的阻碍，其中许多糖基酶是膜蛋白，无法在异源宿主中表达。在这里，我们描述了一种将膜结合糖基转移酶（GTs）拓扑转化为水溶性生物催化剂的策略，该策略在活细胞的细胞质中以高表达水平保留生物活性。我们通过在几种常用的表达平台上轻松生产 98 种难以表达的 GTs（主要来自人类），证明了该方法的通用性。使用这些水溶性酶中的一部分，我们对游离和蛋白连接的聚糖进行结构重塑，包括单克隆抗体治疗药物曲妥珠单抗上的聚糖。总的来说，我们对 GTs 进行合理重新设计的策略为大量不同的、具有酶活性的 GTs 提供了一种有效且通用的生物合成途径，这些 GTs 应该在结构-功能研究以及涉及复杂糖分子的生化和生物医学应用中得到应用。
