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

Liang Lin1,2,3, Weston Kightlinger2,4, Katherine F Warfel2,4

  • 1Department of Biomedical Engineering, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States.

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Summary

Researchers developed a new method to engineer glycosyltransferases for precise protein modification. This advances synthetic glycosylation for protein therapeutics, improving their efficacy and expanding glycoengineering applications.

Keywords:
cell-free protein synthesisglycosylationglycosyltransferasehigh-throughputsynthetic biologytherapeutics

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Protein Engineering

Background:

  • Protein glycosylation is crucial for therapeutic protein activity and efficacy.
  • Current methods for site-specific protein glycosylation are limited, especially in vitro and in bacteria.
  • Engineering glycosyltransferases is key to overcoming these limitations.

Purpose of the Study:

  • To develop an efficient and systematic method for screening glycosyltransferases.
  • To engineer a prokaryotic N-glycosyltransferase (NGT) for broad substrate specificity.
  • To expand the scope of glycoengineering for protein therapeutics.

Main Methods:

  • Utilized cell-free protein synthesis and mass spectrometry of self-assembled monolayers.
  • Screened 26 pools of site-saturated NGT libraries against 1052 unique peptides.
  • Characterized 122 NGT mutants under 52,894 reaction conditions.

Main Results:

  • Identified key residues determining polypeptide specificity for NGTs.
  • Defined a panel of 14 NGTs modifying 93% of canonical eukaryotic glycosylation sequences.
  • Developed a panel for noncanonical sequences, including those with non-S/T amino acids at X+2.
  • Successfully applied engineered NGTs to enhance glycosylation efficiency in three protein therapeutics.

Conclusions:

  • The developed screening method enables efficient engineering of glycosyltransferases.
  • The engineered NGT panels significantly broaden substrate specificity for glycoengineering.
  • This work advances in vitro and bacterial glycoengineering, with implications for protein therapeutic development.