The emerging discipline of bacterial glycoengineering has made it possible to produce designer glycans and glycoconjugates for use as vaccines and therapeutics. Unfortunately, cell-based production of homogeneous glycoproteins remains a significant challenge due to cell viability constraints and the inability to control glycosylation components at precise ratios in vivo. To address these challenges, we describe a novel cell-free glycoprotein synthesis (CFGpS) technology that seamlessly integrates protein biosynthesis with asparagine-linked protein glycosylation. This technology leverages a glyco-optimized
Cell-free systems for accelerating glycoprotein expression and biomanufacturing
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.
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- Award ID(s):
- 1936789
- NSF-PAR ID:
- 10249265
- Date Published:
- Journal Name:
- Journal of Industrial Microbiology and Biotechnology
- Volume:
- 47
- Issue:
- 11
- ISSN:
- 1367-5435
- Page Range / eLocation ID:
- 977 to 991
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Escherichia coli strain to source cell extracts that are selectively enriched with glycosylation components, including oligosaccharyltransferases (OSTs) and lipid-linked oligosaccharides (LLOs). The resulting extracts enable a one-pot reaction scheme for efficient and site-specific glycosylation of target proteins. The CFGpS platform is highly modular, allowing the use of multiple distinct OSTs and structurally diverse LLOs. As such, we anticipate CFGpS will facilitate fundamental understanding in glycoscience and make possible applications in on demand biomanufacturing of glycoproteins. -
Severe Acute respiratory syndrome coronavirus (SARS-CoV-1) attaches to the host cell surface to initiate the interaction between the receptor-binding domain (RBD) of its spike glycoprotein (S) and the human Angiotensin-converting enzyme (hACE2) receptor. SARS-CoV-1 mutates frequently because of its RNA genome, which challenges the antiviral development. Here, we per-formed computational saturation mutagenesis of the S protein of SARS-CoV-1 to identify the residues crucial for its functions. We used the structure-based energy calculations to analyze the effects of the missense mutations on the SARS-CoV-1 S stability and the binding affinity with hACE2. The sequence and structure alignment showed similarities between the S proteins of SARS-CoV-1 and SARS-CoV-2. Interestingly, we found that target mutations of S protein amino acids generate similar effects on their stabilities between SARS-CoV-1 and SARS-CoV-2. For example, G839W of SARS-CoV-1 corresponds to G857W of SARS-CoV-2, which decrease the stability of their S glycoproteins. The viral mutation analysis of the two different SARS-CoV-1 isolates showed that mutations, T487S and L472P, weakened the S-hACE2 binding of the 2003–2004 SARS-CoV-1 isolate. In addition, the mutations of L472P and F360S destabilized the 2003–2004 viral isolate. We further predicted that many mutations on N-linked glycosylation sites would increase the stability of the S glycoprotein. Our results can be of therapeutic importance in the design of antivirals or vaccines against SARS-CoV-1 and SARS-CoV-2.more » « less
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null (Ed.)Abstract Cell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for biomanufacturing and synthetic biology. However, activating membrane-dependent functionality of cell-derived vesicles in bacterial CFE systems has been limited. Here, we address this limitation by characterizing native membrane vesicles in Escherichia coli- based CFE extracts and describing methods to enrich vesicles with heterologous, membrane-bound machinery. As a model, we focus on bacterial glycoengineering. We first use multiple, orthogonal techniques to characterize vesicles and show how extract processing methods can be used to increase concentrations of membrane vesicles in CFE systems. Then, we show that extracts enriched in vesicle number also display enhanced concentrations of heterologous membrane protein cargo. Finally, we apply our methods to enrich membrane-bound oligosaccharyltransferases and lipid-linked oligosaccharides for improving cell-free N- linked and O -linked glycoprotein synthesis. We anticipate that these methods will facilitate on-demand glycoprotein production and enable new CFE systems with membrane-associated activities.more » « less
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Summary The key technical bottleneck for exploiting plant hairy root cultures as a robust bioproduction platform for therapeutic proteins has been low protein productivity, particularly low secreted protein yields. To address this, we engineered novel hydroxyproline (Hyp)‐
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1007/s10295-020-02321-4
