ABSTRACT 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

applicable method to facilitate fundamental understanding of glycosyltransferases and engineer synthetic glycoproteins. Access through your institution Buy or subscribe This is a preview of

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* Log in * Learn about institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS A UNIVERSAL GLYCOENZYME BIOSYNTHESIS PIPELINE THAT

ENABLES EFFICIENT CELL-FREE REMODELING OF GLYCANS Article Open access 24 October 2022 RESTORING PROTEIN GLYCOSYLATION WITH GLYCOSHAPE Article Open access 14 October 2024 AN EFFICIENT

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references ACKNOWLEDGEMENTS 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). AUTHOR INFORMATION Author notes * These authors contributed equally: Weston Kightlinger, Liang Lin. AUTHORS AND AFFILIATIONS * Department of Chemical and

Biological Engineering, Northwestern University, Evanston, IL, USA Weston Kightlinger, Milan Mrksich & Michael C. Jewett * Center for Synthetic Biology, Northwestern University,

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

Rosztoczy, Wenhao Li & Milan Mrksich * Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, USA Matthew P. DeLisa * Department of

Microbiology, Cornell University, Ithaca, NY, USA Matthew P. DeLisa * Department of Chemistry, Northwestern University, Evanston, IL, USA Milan Mrksich Authors * Weston Kightlinger View

author publications You can also search for this author inPubMed Google Scholar * Liang Lin View author publications You can also search for this author inPubMed Google Scholar * Madisen

Rosztoczy View author publications You can also search for this author inPubMed Google Scholar * Wenhao Li View author publications You can also search for this author inPubMed Google

Scholar * Matthew P. DeLisa View author publications You can also search for this author inPubMed Google Scholar * Milan Mrksich View author publications You can also search for this author

inPubMed Google Scholar * Michael C. Jewett View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS 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. CORRESPONDING AUTHORS Correspondence to Milan Mrksich or Michael C. Jewett. ETHICS

DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PUBLISHER’S NOTE: Springer Nature remains neutral with regard to jurisdictional claims in

published maps and institutional affiliations. SUPPLEMENTARY INFORMATION SUPPLEMENTARY TEXT AND FIGURES Supplementary Tables 1–5, Supplementary Figures 1–27, Supplementary Note 1 REPORTING

SUMMARY RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Kightlinger, W., Lin, L., Rosztoczy, M. _et al._ Design of glycosylation sites by rapid synthesis

and analysis of glycosyltransferases. _Nat Chem Biol_ 14, 627–635 (2018). https://doi.org/10.1038/s41589-018-0051-2 Download citation * Received: 24 October 2017 * Accepted: 07 March 2018 *

Published: 07 May 2018 * Issue Date: June 2018 * DOI: https://doi.org/10.1038/s41589-018-0051-2 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this

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