RT Journal Article
SR Electronic
T1 A cell-free biosynthesis platform for modular construction of protein glycosylation pathways
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 833806
DO 10.1101/833806
A1 Weston Kightlinger
A1 Katherine E. Duncker
A1 Ashvita Ramesh
A1 Ariel H. Thames
A1 Aravind Natarajan
A1 Allen Yang
A1 Jessica C. Stark
A1 Liang Lin
A1 Milan Mrksich
A1 Matthew P. DeLisa
A1 Michael C. Jewett
YR 2019
UL http://biorxiv.org/content/early/2019/11/07/833806.abstract
AB Glycosylation plays important roles in cellular function and endows protein therapeutics with beneficial properties. However, constructing biosynthetic pathways to study and engineer protein glycosylation remains a bottleneck. To address this limitation, we describe a modular, versatile cell-free platform for glycosylation pathway assembly by rapid in vitro mixing and expression (GlycoPRIME). In GlycoPRIME, crude cell lysates are enriched with glycosyltransferases by cell-free protein synthesis and then glycosylation pathways are assembled in a mix-and-match fashion to elaborate a single glucose priming handle installed by an N-linked glycosyltransferase. We demonstrate GlycoPRIME by constructing 37 putative protein glycosylation pathways, creating 23 unique glycan motifs. We then use selected pathways to design a one-pot cell-free system to synthesize a vaccine protein with an α-galactose motif and engineered Escherichia coli strains to produce human antibody constant regions with minimal sialic acid motifs. We anticipate that our work will facilitate glycoscience and make possible new glycoengineering applications.