Ribosome Stalling of N-Linked Glycoproteins in Cell-Free Extracts.
Ribosome Stalling of N-Linked Glycoproteins in Cell-Free Extracts.
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DOI：
10.1021/acssynbio.2c00311
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发表时间：
2022-12-16
影响因子：
4.7
通讯作者：
DeLisa, Matthew P.
中科院分区：
文献类型：
--
作者：
Chung, Sean S.;Bidstrup, Erik J.;Hershewe, Jasmine M.;Warfel, Katherine F.;Jewett, Michael C.;DeLisa, Matthew P.
关键词：
核糖体展示是从组合文库中筛选和定向进化蛋白质的一种强大的体外方法。然而，通过糖基化等复杂的翻译后修饰来展示蛋白质的能力是有限的。为了弥补这一差距，我们开发了一套补充方法来生产停滞的核糖体复合体，这些复合体显示天冬酰胺连接(N连接)的糖蛋白，其构象服从下游功能和糖结构的询问。通过将SECM介导的翻译抑制与无细胞N-糖蛋白合成方法相结合，能够产生糖基化核糖体-新生链复合体(GRNC)。这种整合实现了一种首创的方法，可以用不同的N-糖链结构高效地定点修饰目标蛋白的核糖体停滞。此外，观察到编码的mRNAs仍然稳定地附着在核糖体上，这为阻止糖蛋白与其RNA信息之间的基因-糖型联系提供了证据。我们预计，我们的方法将能够选择和进化具有良好生物学和生物物理特性的N-糖蛋白。
Ribosome display is a powerful in vitro method for selection and directed evolution of proteins expressed from combinatorial libraries. However, the ability to display proteins with complex post-translational modifications such as glycosylation is limited. To address this gap, we developed a set of complementary methods for producing stalled ribosome complexes that displayed asparagine-linked (N-linked) glycoproteins in conformations amenable to downstream functional and glycostructural interrogation. The ability to generate glycosylated ribosome–nascent chain (glycoRNC) complexes was enabled by integrating SecM-mediated translation arrest with methods for cell-free N-glycoprotein synthesis. This integration enabled a first-in-kind method for ribosome stalling of target proteins modified efficiently and site-specifically with different N-glycan structures. Moreover, the observation that encoding mRNAs remained stably attached to ribosomes provides evidence of a genotype–glycophenotype link between an arrested glycoprotein and its RNA message. We anticipate that our method will enable selection and evolution of N-glycoproteins with advantageous biological and biophysical properties.
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影响因子：
8
作者：
Celik, Eda;Fisher, Adam C.;DeLisa, Matthew P.
通讯作者：
DeLisa, Matthew P.
影响因子：
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影响因子：
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作者：
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通讯作者：
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