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.
Document Details
- Document Type
- Pub Defense Publication
- Publication Date
- Nov 27, 2019
- Source ID
- 10.1038/s41467-019-12024-9
Entities
People
- Allen Yang
- Aravind Natarajan
- Ariel H Thames
- Ashvita Ramesh
- Jessica C Stark
- Katherine E Duncker
- Liang Lin
- Matthew P. DeLisa
- Michael C Jewett
- Milan Mrksich
- Weston Kightlinger
Organizations
- David and Lucile Packard Foundation
- Defense Threat Reduction Agency
- National Science Foundation
- The Camille and Henry Dreyfus Foundation