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

Thapakorn Jaroentomeechai, Jessica C. Stark, Aravind Natarajan, Cameron J. Glasscock, Laura E. Yates, Karen J. Hsu, Milan Mrksich, Michael C. Jewett, Matthew P. Delisa*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

134 Scopus citations

Abstract

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.

Original languageEnglish (US)
Article number2686
JournalNature communications
Volume9
Issue number1
DOIs
StatePublished - Dec 1 2018

ASJC Scopus subject areas

  • General Physics and Astronomy
  • General Chemistry
  • General Biochemistry, Genetics and Molecular Biology

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