A cell-free platform for rapid synthesis and testing of active oligosaccharyltransferases

Jennifer A. Schoborg, Jasmine M. Hershewe, Jessica C. Stark, Weston Kightlinger, James E. Kath, Thapakorn Jaroentomeechai, Aravind Natarajan, Matthew P. DeLisa, Michael C. Jewett*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

32 Scopus citations

Abstract

Protein glycosylation, or the attachment of sugar moieties (glycans) to proteins, is important for protein stability, activity, and immunogenicity. However, understanding the roles and regulations of site-specific glycosylation events remains a significant challenge due to several technological limitations. These limitations include a lack of available tools for biochemical characterization of enzymes involved in glycosylation. A particular challenge is the synthesis of oligosaccharyltransferases (OSTs), which catalyze the attachment of glycans to specific amino acid residues in target proteins. The difficulty arises from the fact that canonical OSTs are large (>70 kDa) and possess multiple transmembrane helices, making them difficult to overexpress in living cells. Here, we address this challenge by establishing a bacterial cell-free protein synthesis platform that enables rapid production of a variety of OSTs in their active conformations. Specifically, by using lipid nanodiscs as cellular membrane mimics, we obtained yields of up to 420 μg/ml for the single-subunit OST enzyme, “Protein glycosylation B” (PglB) from Campylobacter jejuni, as well as for three additional PglB homologs from Campylobacter coli, Campylobacter lari, and Desulfovibrio gigas. Importantly, all of these enzymes catalyzed N-glycosylation reactions in vitro with no purification or processing needed. Furthermore, we demonstrate the ability of cell-free synthesized OSTs to glycosylate multiple target proteins with varying N-glycosylation acceptor sequons. We anticipate that this broadly applicable production method will advance glycoengineering efforts by enabling preparative expression of membrane-embedded OSTs from all kingdoms of life.

Original languageEnglish (US)
Pages (from-to)739-750
Number of pages12
JournalBiotechnology and Bioengineering
Volume115
Issue number3
DOIs
StatePublished - Mar 2018

Keywords

  • PglB
  • asparagine-linked protein glycosylation
  • cell-free protein synthesis
  • membrane protein
  • nanodisc
  • oligosaccharyltransferase
  • post-translational modification
  • synthetic biology

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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