Directed Evolution of Glycopeptides Using mRNA Display

Abstract

Directed evolution is a useful method for the discovery of nucleic acids, peptides or proteins that have desired binding abilities or functions. Because of the abundance and importance of glycosylation in nature, directed evolution of glycopeptides and glycoproteins is also highly desirable. However, common directed evolution platforms such as phage-, yeast-, or mammalian-cell display are limited for these applications by several factors. Glycan structure at each glycosylation site is not genetically encoded, and yeast and mammalian cells produce a heterogenous mixture of glycoforms at each site on the protein. Although yeast, mammalian and Escherichia coli cells can be engineered to produce a homogenous glycoform at all glycosylation sites, there are just a few specific glycan structures that can readily be accessed in this manner. Recently, we reported a novel system for the directed evolution of glycopeptide libraries, which could in principle be decorated with any desired glycan. Our method combines in vitro peptide selection by mRNA display with unnatural amino acid incorporation and chemical attachment of synthetic oligosaccharides. Here, we provide an updated and optimized protocol for this method, which is designed to create glycopeptide mRNA display libraries containing ~1013 sequences and select them for target binding. The target described here is the HIV broadly-neutralizing monoclonal antibody 2G12; 2G12 binds to cluster of high-mannose oligosaccharides on the HIV envelope glycoprotein gp120 and glycopeptides that mimic this epitope may be useful in HIV vaccine applications. This method is expected to be readily applicable for other types of glycans and targets of interest in glycobiology.

Decentralizing Cell-Free RNA Sensing With the Use of Low-Cost Cell Extracts

“…Furthermore, they need to be imported from the Northern hemisphere under cold-chain shipping, which increases their costs and the risk of activity loss. PURE systems can also be prepared in the laboratory by purifying the components individually or as a single step from a bacterial consortium of strains ( Shimizu et al, 2001 ; Horiya et al, 2017 ; Villarreal et al, 2018 ; Lavickova and Maerkl, 2019 ). Alternatively, cell lysate-based protein expression systems, made from E. coli crude extract that do not require purification of particular components, can also be used as a substrate for CFTS ( Silverman et al, 2019 ).…”

Oligomannose Glycopeptide Conjugates Elicit Antibodies Targeting the Glycan Core Rather than Its Extremities

“…Our laboratory has created directed evolution-based techniques for design of glycan clusters that may mimic such epitopes (Figure 1b), enabling us to discover structures with high antigenicity (low nM K D ) for 2G12. 41−46 Herein, we report structural studies and immunogenicity of these synthetically glycosylated peptides in rabbits.…”

Bottom-Up Construction of Complex Biomolecular Systems With Cell-Free Synthetic Biology

“…Cost-effective and modular PURE systems with user-defined compositions can be prepared in the laboratory (Shimizu and Ueda, 2010;Horiya et al, 2017), but the labor-intensive protocol requires ∼36 medium to large scale His-tag and ribosome purification steps (Figure 2A). Thus, different approaches to simplify the protocol have been developed, including Histagging of in vivo enzyme pathways (Wang et al, 2012), microbial consortia (Villarreal et al, 2018), and bacterial artificial chromosomes (Shepherd et al, 2017).…”