Cell-free systems for accelerating glycoprotein expression and biomanufacturing
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METABOLIC ENGINEERING AND SYNTHETIC BIOLOGY - REVIEW
Cell‑free systems for accelerating glycoprotein expression and biomanufacturing Jasmine Hershewe1,2,3 · Weston Kightlinger1,2,3 · Michael C. Jewett1,2,3,4,5 Received: 9 July 2020 / Accepted: 3 October 2020 © Society for Industrial Microbiology and Biotechnology 2020
Abstract Protein glycosylation, the enzymatic modification of amino acid sidechains with sugar moieties, plays critical roles in cellular function, human health, and biotechnology. However, studying and producing defined glycoproteins remains challenging. Cell-free glycoprotein synthesis systems, in which protein synthesis and glycosylation are performed in crude cell extracts, offer new approaches to address these challenges. Here, we review versatile, state-of-the-art systems for biomanufacturing glycoproteins in prokaryotic and eukaryotic cell-free systems with natural and synthetic N-linked glycosylation pathways. We discuss existing challenges and future opportunities in the use of cell-free systems for the design, manufacture, and study of glycoprotein biomedicines. Keywords Cell-free protein synthesis · Glycosylation · Glycoengineering · High-throughput experimentation · Synthetic biology Abbreviations CFGpS Cell-free glycoprotein synthesis CFPS Cell-free protein synthesis CHO Chinese hamster ovary Cj Campylobacter jejuni DSB Disulfide bond EGFR Epidermal growth factor receptor ELISA Enzyme-linked immunosorbent assay EPO Erythropoietin * Michael C. Jewett m‑[email protected] 1
Department of Chemical and Biological Engineering, Northwestern University, Technological Institute E136, 2145 Sheridan Road, Evanston, IL 60208–3120, USA
2
Chemistry of Life Processes Institute, Northwestern University, 2170 Campus Drive, Evanston, IL 60208–3120, USA
3
Center for Synthetic Biology, Northwestern University, Technological Institute E136, 2145 Sheridan Road, Evanston, IL 60208–3120, USA
4
Robert H. Lurie Comprehensive Cancer Center, Northwestern University, 676 North Saint Clair Street, Suite 1200, Chicago, IL 60611–3068, USA
5
Simpson Querrey Institute, Northwestern University, 303 East Superior Street, Suite 11‑131, Chicago, IL 60611–2875, USA
ER Endoplasmic reticulum Ft Francisella tularensis Gal Galactose GalNAc N-acetyl galactosamine Glc Glucose GlcNAc N-acetyl glucosamine GlycoPRIME Glycosylation pathway assembly by rapid in vitro mixing and expression GlycoSCORES Glycosylation sequence characterization and optimization by rapid expression and screening GOx Glucose oxidase gp120 HIV-1 envelope glycoprotein GT Glycosyltransferase IgG Immunoglobulin G IRES Internal ribosome mediated entry site iVAX In vitro bioconjugate vaccine expression LLO Lipid-linked oligosaccharide Luc Firefly luciferase Man Mannose MBP Maltose binding protein MS Mass spectrometry N-linked glycosylation Asparagine-linked glycosylation ncAA Non-canonical amino acid
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Vol.:(0123456789)
NGT N-Glycosyltransferase O-linked glycosylation Serine/ threonine-li
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