A universal glycoenzyme biosynthesis pipeline that enables efficient cell-free remodeling of glycans.
A universal glycoenzyme biosynthesis pipeline that enables efficient cell-free remodeling of glycans.
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DOI：
10.1038/s41467-022-34029-7
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发表时间：
2022-10-24
影响因子：
16.6
通讯作者：
中科院分区：
文献类型：
--
作者：
重建天然糖基化途径或从头开始构建全新途径的能力受到功能性糖酶可用性的限制，其中许多是膜蛋白，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，这种生物催化剂在活细胞的细胞质中高水平表达，并保持生物活性。我们通过在几种常用的表达平台上轻松生成98个难以表达的GTs（主要来自人类），证明了该方法的普遍性。使用这些水溶性酶的一个子集，我们对游离聚糖和蛋白连接聚糖进行结构重塑，包括在单克隆抗体治疗曲妥珠单抗上发现的那些聚糖。总之，我们合理重新设计GTs的策略为大量不同的酶活性GTs提供了有效和通用的生物合成途径，这应该在结构-功能研究以及涉及复杂糖分子的生化和生物医学应用中找到用途。用于基础和应用研究的糖酶的获取受到重组表达困难的限制。在这里，作者描述了一种将膜结合的糖基转移酶转化为水溶性生物催化剂的通用策略，这种生物催化剂在高水平上表达并保持活性。
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. Access to glycoenzymes for basic and applied research is limited by difficulties with their recombinant expression. Here, the authors describe a universal strategy for converting membrane-bound glycosyltransferases into water-soluble biocatalysts, which are expressed at high levels with retention of activity.
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影响因子：
14.8
作者：
Gietz, R. Daniel;Schiestl, Robert H.
通讯作者：
Schiestl, Robert H.
影响因子：
3.8
作者：
Jewett, MC;Swartz, JR
通讯作者：
Swartz, JR
影响因子：
8.4
作者：
Karim, Ashty S.;Jewett, Michael C.
通讯作者：
Jewett, Michael C.
DOI：
10.1073/pnas.1716988115
发表时间：
2018-05-01
影响因子：
11.1
作者：
Kadirvelraj, Renuka;Yang, Jeong-Yeh;Moremen, Kelley W.
通讯作者：
Moremen, Kelley W.
影响因子：
3
作者：
Hidari, KIPJ;Horie, N;Suzuki, Y
通讯作者：
Suzuki, Y