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ACS Synth Biol ; 11(12): 3892-3899, 2022 12 16.
Article in English | MEDLINE | ID: mdl-36399685

ABSTRACT

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.


Subject(s)
Protein Biosynthesis , Ribosomes , Cell Extracts , Ribosomes/genetics , Ribosomes/metabolism , RNA, Messenger/metabolism , Glycoproteins/genetics , Glycoproteins/metabolism
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