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

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 Nglycoprotein 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.

The SecM arrest peptide traps a pre-peptide bond formation state of the ribosome

“…In addition to studies addressing the mechanism of SecM-mediated stalling, the SecM arrest peptide has been used extensively for generating ribosome-nascent chain complexes (RNCs) for functional studies 28 – 30 , including ribosome display 31 , 32 , real-time monitoring in vivo 33 and single molecule imaging 34 , 35 , but particularly for investigating co-translational protein folding and targeting events 36 – 53 . Furthermore, molecular dynamics simulations based on available structural models for SecM have been performed to investigate how the pulling force could relieve the translational arrest 38 , 54 – 56 .…”

Carbohydrate-active enzyme (CAZyme) discovery and engineering via (Ultra)high-throughput screening

“…128,129 Phage and mRNA display offer new pathways for determining sequence preferences and engineering bacterial Nglycosylation. 130,131 In 2010, the Aebi group developed a method known as ''GlycoPhage'' (Fig. 8c).…”

“…131 The polypeptide can then be glycosylated through cell-free glycoprotein synthesis, the glycoprotein enriched by affinity purification, and the sequence of the RNA within the stalled ribosome determined by sequencing of the RT-PCR product. 131 While not described within the paper, this method should be applicable to engineering/determination of glycosylation sequons and other aspects of protein glycosylation and affords a much higher throughput than cell-based methods (410 9 clones per screen). 131 A number of studies have applied self-assembled monolayers for matrix-assisted desorption/ionization mass spectrometry (SAMDI-MS) for determination of sequence specificities (Fig.…”

“…131 While not described within the paper, this method should be applicable to engineering/determination of glycosylation sequons and other aspects of protein glycosylation and affords a much higher throughput than cell-based methods (410 9 clones per screen). 131 A number of studies have applied self-assembled monolayers for matrix-assisted desorption/ionization mass spectrometry (SAMDI-MS) for determination of sequence specificities (Fig. 8e).…”

“…8d ). 131 In this method, the protein of interest is fused to a short sequence which stalls the ribosome such that the nascent polypeptide and mRNA are still bound within the ribosome. 131 The polypeptide can then be glycosylated through cell-free glycoprotein synthesis, the glycoprotein enriched by affinity purification, and the sequence of the RNA within the stalled ribosome determined by sequencing of the RT-PCR product.…”