Glycosylation is an abundant post-translational modification that is important in disease and biotechnology. Current methods to understand and engineer glycosylation cannot sufficiently explore the vast experimental landscapes required to accurately predict and design glycosylation sites modified by glycosyltransferases. Here we describe a systematic platform for glycosylation sequence characterization and optimization by rapid expression and screening (GlycoSCORES), which combines cell-free protein synthesis and mass spectrometry of self-assembled monolayers. We produced six N- and O-linked polypeptide-modifying glycosyltransferases from bacteria and humans in vitro and rigorously determined their substrate specificities using 3,480 unique peptides and 13,903 unique reaction conditions. We then used GlycoSCORES to optimize and design small glycosylation sequence motifs that directed efficient N-linked glycosylation in vitro and in the Escherichia coli cytoplasm for three heterologous proteins, including the human immunoglobulin Fc domain. We find that GlycoSCORES is a broadly applicable method to facilitate fundamental understanding of glycosyltransferases and engineer synthetic glycoproteins.

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The authors acknowledge J.C. Stark and J. Hershewe for assistance with western blotting, helpful discussions, and sharing of reagents and ideas; S. Habibi for assistance with LC-TOF instrumentation; and A. Karim for helpful conversations. The authors also thank J. Kath for supply of plasmids, advice on protein expression, and critical reading of the manuscript. We also thank A. Natarajan of the Department of Microbiology at Cornell University, T. Jaroentomeechai of the Robert Frederick Smith School of Chemical and Biomolecular Engineering at Cornell University, and J. Janetzko of the Department of Chemistry and Chemical Biology at Harvard University for sharing the ppGalNAcT, Im7, and hOGT source plasmids, respectively. This work made use of the Integrated Molecular Structure Education and Research Center at Northwestern University, which has received support from the state of Illinois, the Northwestern University Office of Research and the Chemistry Department for LC-TOF instrumentation. This material is based upon work supported by the Defense Threat Reduction Agency (HDTRA1-15-10052/P00001), the David and Lucile Packard Foundation, the Dreyfus Teacher-Scholar program, and the National Science Foundation (Graduate Research Fellowship under Grant No. DGE-1324585 and MCB-1413563).

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Author notes

  1. These authors contributed equally: Weston Kightlinger, Liang Lin.


  1. Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, USA

    • Weston Kightlinger
    • , Milan Mrksich
    •  & Michael C. Jewett
  2. Center for Synthetic Biology, Northwestern University, Evanston, IL, USA

    • Weston Kightlinger
    • , Liang Lin
    • , Milan Mrksich
    •  & Michael C. Jewett
  3. Department of Biomedical Engineering, Northwestern University, Evanston, IL, USA

    • Liang Lin
    • , Madisen Rosztoczy
    • , Wenhao Li
    •  & Milan Mrksich
  4. Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, USA

    • Matthew P. DeLisa
  5. Department of Microbiology, Cornell University, Ithaca, NY, USA

    • Matthew P. DeLisa
  6. Department of Chemistry, Northwestern University, Evanston, IL, USA

    • Milan Mrksich


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W.K. and L.L. designed, performed, and analyzed experiments. M.R. designed and optimized experimental protocols. W.L. helped to synthesize peptide libraries. M.M. and M.C.J. directed the studies and interpreted the data. W.K., L.L., M.P.D., M.M., and M.C.J. conceived of the study and wrote the manuscript with assistance from M.R. and W.L.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Milan Mrksich or Michael C. Jewett.

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