Abstract
In recent years, cell-free synthetic glycobiology technologies have emerged that enable production and remodeling of glycoproteins outside the confines of the cell. However, many of these systems combine multiple synthesis steps into one pot where there can be competing reactions and side products that ultimately lead to low yield of the desired product. In this work, we describe a microfluidic platform that integrates cell-free protein synthesis, glycosylation, and purification of a model glycoprotein in separate compartments where each step can be individually optimized. Microfluidics offer advantages such as reaction compartmentalization, tunable residence time, the ability to tether enzymes for reuse, and the potential for continuous manufacturing. Moreover, it affords an opportunity for spatiotemporal control of glycosylation reactions that is difficult to achieve with existing cell-based and cell-free glycosylation systems. In this work, we demonstrate a flow-based glycoprotein synthesis system that promotes enhanced cell-free protein synthesis, efficient protein glycosylation with an immobilized oligosaccharyltransferase, and enrichment of the protein product from cell-free lysate. Overall, this work represents a first-in-kind glycosylation-on-a-chip prototype that could find use as a laboratory tool for mechanistic dissection of the protein glycosylation process as well as a biomanufacturing platform for small batch, decentralized glycoprotein production.
Highlights
Protein glycosylation is a major posttranslational modification where complex carbohydrates known as glycans are enzymatically added to amino acid sidechains of a protein at specific, regioselective positions
In the first module of the device, superfolder green fluorescent protein (sfGFP) bearing a C-terminal DQNAT glycosylation motif (Fisher et al, 2011) that is optimally recognized by Campylobacter jejuni OST enzyme PglB (CjPglB) (Kowarik et al, 2006a; Gerber et al, 2013) and a hexahistidine tag was expressed using crude S30 extract derived from E. coli, which enabled transcription and translation of the target protein on chip
In the third module, the sfGFPDQNAT−6xHis product was selectively enriched using a microfluidic device loaded with affinity resin that facilitated reversible capture of the hexahistidine-tagged glycoprotein product
Summary
Protein glycosylation is a major posttranslational modification where complex carbohydrates known as glycans are enzymatically added to amino acid sidechains of a protein at specific, regioselective positions. The attachment of glycans to asparagine residues, known as N-linked glycosylation, is the most abundant type of glycosylation and occurs in all domains of life (Abu-Qarn et al, 2008) This mode of glycosylation gives rise to diverse chemical structures that are well known to affect the biological and biophysical properties of a protein (Imperiali and O’Connor, 1999; Wolfert and Boons, 2013; Hebert et al, 2014; Lin et al, 2015). Because of these pronounced effects, there is a strong incentive to study glycosylation and leverage the resulting knowledge for the development of glycoengineered proteins with advantageous. There remains a need for alternative methods to produce structurally uniform glycans in sufficient quantities for mechanistic studies and other downstream applications
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