Single-pot glycoprotein biosynthesis using a cell-free transcription-translation system enriched with glycosylation machinery

• Published in: Nature communications 2018-07, Vol.9 (1), p.2686-11, Article 2686
• Main Authors: Jaroentomeechai, Thapakorn, Stark, Jessica C., Natarajan, Aravind, Glasscock, Cameron J., Yates, Laura E., Hsu, Karen J., Mrksich, Milan, Jewett, Michael C., DeLisa, Matthew P.
• Format: Article
• Language: English
• Subjects: 38/1; 38/35; 38/44; 631/45/221; 631/61/185; 631/92/458; 631/92/552; 631/92/60; 82/29; 82/58; 82/80; 82/83; Asparagine; Biosynthesis; Biotechnology - methods; Cell viability; E coli; Escherichia coli; Escherichia coli - genetics; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Glycoconjugates; Glycoproteins; Glycoproteins - genetics; Glycosylation; Hexosyltransferases - genetics; Hexosyltransferases - metabolism; Humanities and Social Sciences; Lipids; Lipopolysaccharides - metabolism; Membrane Proteins - genetics; Membrane Proteins - metabolism; multidisciplinary; Oligosaccharides; Polysaccharides; Protein biosynthesis; Protein Biosynthesis - genetics; Proteins; Reproducibility of Results; Science; Science (multidisciplinary); Transcription; Transcription, Genetic - genetics; Vaccines
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-018-05110-x

The emerging discipline of bacterial glycoengineering has made it possible to produce designer glycans and glycoconjugates for use as vaccines and therapeutics. Unfortunately, cell-based production of homogeneous glycoproteins remains a significant challenge due to cell viability constraints and the inability to control glycosylation components at precise ratios in vivo. To address these challenges, we describe a novel cell-free glycoprotein synthesis (CFGpS) technology that seamlessly integrates protein biosynthesis with asparagine-linked protein glycosylation. This technology leverages a glyco-optimized Escherichia coli strain to source cell extracts that are selectively enriched with glycosylation components, including oligosaccharyltransferases (OSTs) and lipid-linked oligosaccharides (LLOs). The resulting extracts enable a one-pot reaction scheme for efficient and site-specific glycosylation of target proteins. The CFGpS platform is highly modular, allowing the use of multiple distinct OSTs and structurally diverse LLOs. As such, we anticipate CFGpS will facilitate fundamental understanding in glycoscience and make possible applications in on demand biomanufacturing of glycoproteins. The ability to produce homogeneous glycoproteins is expected to advance fundamental understanding in glycoscience, but current in vivo-based production systems have several limitations. Here, the authors develop an E. coli extract-based one-pot system for customized production of N -linked glycoproteins.