Using High-Throughput Experiments To Screen N-Glycosyltransferases with Altered Specificities

Liang Lin, Weston Kightlinger, Katherine F. Warfel, Michael C. Jewett*, Milan Mrksich*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

Abstract

The important roles that protein glycosylation plays in modulating the activities and efficacies of protein therapeutics have motivated the development of synthetic glycosylation systems in living bacteria and in vitro. A key challenge is the lack of glycosyltransferases that can efficiently and site-specifically glycosylate desired target proteins without the need to alter primary amino acid sequences at the acceptor site. Here, we report an efficient and systematic method to screen a library of glycosyltransferases capable of modifying comprehensive sets of acceptor peptide sequences in parallel. This approach is enabled by cell-free protein synthesis and mass spectrometry of self-assembled monolayers and is used to engineer a recently discovered prokaryotic N-glycosyltransferase (NGT). We screened 26 pools of site-saturated NGT libraries to identify relevant residues that determine polypeptide specificity and then characterized 122 NGT mutants, using 1052 unique peptides and 52,894 unique reaction conditions. We define a panel of 14 NGTs that can modify 93% of all sequences within the canonical X-1-N-X+1-S/T eukaryotic glycosylation sequences as well as another panel for many noncanonical sequences (with 10 of 17 non-S/T amino acids at the X+2 position). We then successfully applied our panel of NGTs to increase the efficiency of glycosylation for three protein therapeutics. Our work promises to significantly expand the substrates amenable to in vitro and bacterial glycoengineering.

Original languageEnglish (US)
Pages (from-to)1290-1302
Number of pages13
JournalACS synthetic biology
Volume13
Issue number4
DOIs
StatePublished - Apr 19 2024

Funding

This work was supported by the Defense Threat Reduction Agency (HDTRA1-21-1-0038, HDTRA1-20-1-0004), DARPA (W911NF-23-2-0039), and the National Science Foundation Graduate Research Fellowship program (DGE-1324585). We acknowledge Andrew Ott for assistance with LC-qTOF instrumentation. This work made use of IMSERC at Northwestern University, which has received support from the Soft and Hybrid Nanotechnology Experimental (SHyNE) Resource (NSF ECCS-1542205), the State of Illinois and International Institute for Nanotechnology (IIN).

Keywords

  • cell-free protein synthesis
  • glycosylation
  • glycosyltransferase
  • high-throughput
  • synthetic biology
  • therapeutics

ASJC Scopus subject areas

  • Biomedical Engineering
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)

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