Established and Emerging Methods for Protecting Linear DNA in Cell-Free Expression Systems

Fochtman, Trevor J.; Oza, Javin P.

doi:10.3390/mps6020036

Cited by 4 publications

(3 citation statements)

References 54 publications

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“…The Solid-State Biologics platform’s ability to support complex biosynthesis utilizing a mixture of bioreagents in a variety of reaction formats demonstrates its versatility for rapid and on-demand applications where current infrastructure remains limiting. Examples range from R&D and diagnostics to biomanufacturing of commodity chemicals and therapeutics in resource limited environments. ,, The observed results also demonstrate potential for integration into the classroom, where CFPS has a the potential to improve learning outcomes in biotechnology workforce development efforts. , Lastly, SSBs provide a foundation for extending the shelf-life of biologics. Specifically, previous work on integrating excipients into lyophilized biologics have successfully improved shelf-life by weeks to months. ,,,− Having included beneficial excipients into SSBs, we anticipate similar gains in shelf-life.…”

Section: Discussionmentioning

confidence: 83%

“…Examples range from R&D and diagnostics to biomanufacturing of commodity chemicals and therapeutics in resource limited environments. 78,127,128 The observed results also demonstrate potential for integration into the classroom, where CFPS has a the potential to improve learning outcomes in biotechnology workforce development efforts. 80,129 Lastly, SSBs provide a foundation for extending the shelf-life of biologics.…”

Section: ■ Conclusionmentioning

confidence: 99%

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Development of Solid-State Storage for Cell-Free Expression Systems

et al. 2023

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The fragility of biological systems during storage, transport, and utilization necessitates reliable cold-chain infrastructure and limits the potential of biotechnological applications. In order to unlock the broad applications of existing and emerging biological technologies, we report the development of a novel solid-state storage platform for complex biologics. The resulting solid-state biologics (SSB) platform meets four key requirements: facile rehydration of solid materials, activation of biochemical activity, ability to support complex downstream applications and functionalities, and compatibility for deployment in a variety of reaction formats and environments. As a model system of biochemical complexity, we utilized crudeEscherichia colicell extracts that retain active cellular metabolism and support robust levels of in vitro transcription and translation. We demonstrate broad versatility and utility of SSB through proof-of-concepts for on-demand in vitro biomanufacturing of proteins at a milliliter scale, the activation of downstream CRISPR activity, as well as deployment on paper-based devices. SSBs unlock a breadth of applications in biomanufacturing, discovery, diagnostics, and education in resource-limited environments on Earth and in space.

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Section: Discussionmentioning

confidence: 83%

Section: ■ Conclusionmentioning

confidence: 99%

Development of Solid-State Storage for Cell-Free Expression Systems

et al. 2023

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“…Further acceleration of BYL workflows are in development to expand upon this potential, such as the application of alternative DNA templates beyond the standard plasmid format. Taking inspiration from E. coli CFPS, there are a variety of strategies that could be trialed for adapting linear DNA templates in BYL (Fochtman & Oza, 2023;Sato et al, 2022;Zhu et al, 2020).…”

Section: Discussionmentioning

confidence: 99%

Scaling eukaryotic cell‐free protein synthesis achieved with the versatile and high‐yielding tobacco BY‐2 cell lysate

Gupta¹,

Flaskamp²,

Roentgen³

et al. 2023

Biotech & Bioengineering

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Eukaryotic cell‐free protein synthesis (CFPS) can accelerate expression and high‐throughput analysis of complex proteins with functionally relevant post‐translational modifications (PTMs). However, low yields and difficulties scaling such systems have prevented their widespread adoption in protein research and manufacturing.Here, we provide detailed demonstrations for the capabilities of a CFPS system derived from Nicotiana tabacum BY‐2 cell culture (BY‐2 lysate; BYL). BYL is able to express diverse, functional proteins at high yields in 48 h, complete with native disulfide bonds and N‐glycosylation. An optimized version of the technology is commercialized as ALiCE® and advances in scaling of BYL production methodologies now allow scaling of eukaryotic CFPS reactions. We show linear, lossless scale‐up of batch mode protein expression from 100 µL microtiter plates to 10 and 100 mL volumes in Erlenmeyer flasks, culminating in preliminary data from a litre‐scale reaction in a rocking‐type bioreactor. Together, scaling across a 20,000x range is achieved without impacting product yields.Production of multimeric virus‐like particles from the BYL cytosolic fraction were then shown, followed by functional expression of multiple classes of complex, difficult‐to‐express proteins using the native microsomes of the BYL CFPS. Specifically: a dimeric enzyme; a monoclonal antibody; the SARS‐CoV‐2 receptor‐binding domain; a human growth factor; and a G protein‐coupled receptor membrane protein. Functional binding and activity are demonstrated, together with in‐depth PTM characterization of purified proteins through disulfide bond and N‐glycan analysis.Taken together, BYL is a promising end‐to‐end R&D to manufacturing platform with the potential to significantly reduce the time‐to‐market for high value proteins and biologics.

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Split Reporters Facilitate Monitoring of Gene Expression and Peptide Production in Linear Cell-Free Transcription–Translation Systems

Lévrier,

Capin,

Mayonove

et al. 2024

ACS Synth. Biol.

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Cell-free transcription−translation (TXTL) systems expressing genes from linear dsDNA enable the rapid prototyping of genetic devices while avoiding cloning steps. However, repetitive inclusion of a reporter gene is an incompressible cost and sometimes accounts for most of the synthesized DNA length. Here we present reporter systems based on split-GFP systems that reassemble into functional fluorescent proteins and can be used to monitor gene expression in E. coli TXTL. The 135 bp GFP10−11 fragment produces a fluorescent signal comparable to its full-length GFP counterpart when reassembling with its complementary protein synthesized from the 535 bp fragment expressed in TXTL. We show that split reporters can be used to characterize promoter libraries, with data qualitatively comparable to full-length GFP and matching in vivo expression measurements. We also use split reporters as small fusion tags to measure the TXTL protein and peptide production yield. Finally, we generalize our concept by providing a luminescent split reporter based on split-nanoluciferase. The ∼80% gene sequence length reduction afforded by split reporters lowers synthesis costs and liberates space for testing larger devices while producing a reliable output. In the peptide production context, the small size of split reporters compared with full-length GFP is less likely to bias peptide solubility assays. We anticipate that split reporters will facilitate rapid and cost-efficient genetic device prototyping, protein production, and interaction assays.

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Established and Emerging Methods for Protecting Linear DNA in Cell-Free Expression Systems

Cited by 4 publications

References 54 publications

Development of Solid-State Storage for Cell-Free Expression Systems

Development of Solid-State Storage for Cell-Free Expression Systems

Scaling eukaryotic cell‐free protein synthesis achieved with the versatile and high‐yielding tobacco BY‐2 cell lysate

Split Reporters Facilitate Monitoring of Gene Expression and Peptide Production in Linear Cell-Free Transcription–Translation Systems

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