@article{28de789a00c54b508c92dc2826d3f712,
title = "A cell-free biosynthesis platform for modular construction of protein glycosylation pathways",
abstract = "Glycosylation plays important roles in cellular function and endows protein therapeutics with beneficial properties. However, constructing biosynthetic pathways to study and engineer precise glycan structures on proteins remains a bottleneck. Here, we report a modular, versatile cell-free platform for glycosylation pathway assembly by rapid in vitro mixing and expression (GlycoPRIME). In GlycoPRIME, glycosylation pathways are assembled by mixing-and-matching cell-free synthesized glycosyltransferases that can elaborate a glucose primer installed onto protein targets by an N-glycosyltransferase. We demonstrate GlycoPRIME by constructing 37 putative protein glycosylation pathways, creating 23 unique glycan motifs, 18 of which have not yet been synthesized on proteins. We use selected pathways to synthesize a protein vaccine candidate with an α-galactose adjuvant motif in a one-pot cell-free system and human antibody constant regions with minimal sialic acid motifs in glycoengineered Escherichia coli. We anticipate that these methods and pathways will facilitate glycoscience and make possible new glycoengineering applications.",
author = "Weston Kightlinger and Duncker, {Katherine E.} and Ashvita Ramesh and Thames, {Ariel H.} and Aravind Natarajan and Stark, {Jessica C.} and Allen Yang and Liang Lin and Milan Mrksich and DeLisa, {Matthew P.} and Jewett, {Michael C.}",
note = "Funding Information: The authors acknowledge T. Jaroentomeechai, A. Karim, J. Hershewe, and J. Kath for helpful critiques and sharing of reagents as well as S. Habibi, A. Ott, and S. Shafie for assistance with LC-MS instrumentation. This work made use of the IMSERC core facility at Northwestern University, which has received support from the Soft and Hybrid Nanotechnology Experimental (SHyNE) Resource (NSF ECCS-1542205), the State of Illinois, and the International Institute for Nanotechnology (IIN). 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, the National Institutes of Health (NIH) and National Institutes of Environmental Health Sciences (NIEHS) through T32 ES007059, and the National Science Foundation through MCB-1413563 and the Graduate Research Fellowship program (DGE-1324585). Its contents are the sole responsibility of the authors and do not necessarily represent the official views the funding agencies above. Publisher Copyright: {\textcopyright} 2019, The Author(s).",
year = "2019",
month = dec,
day = "1",
doi = "10.1038/s41467-019-12024-9",
language = "English (US)",
volume = "10",
journal = "Nature Communications",
issn = "2041-1723",
publisher = "Nature Publishing Group",
number = "1",
}
