Ribosome Stalling of N‑Linked Glycoproteins in Cell-Free Extracts

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...

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Bibliographic Details
Published in:ACS synthetic biology 2022-12, Vol.11 (12), p.3892-3899
Main Authors: Chung, Sean S., Bidstrup, Erik J., Hershewe, Jasmine M., Warfel, Katherine F., Jewett, Michael C., DeLisa, Matthew P.
Format: Article
Language:English
Subjects:
Cell Extracts
Glycoproteins - genetics
Glycoproteins - metabolism
Letter
Protein Biosynthesis
Ribosomes - genetics
Ribosomes - metabolism
RNA, Messenger - metabolism
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Summary: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.
ISSN:2161-5063
2161-5063
DOI:10.1021/acssynbio.2c00311