Advancing synthetic biology through cell-free protein synthesis

Ke, Yanxiong; Chen, Junyu; Li, Yingqiu; Lou, Kai

doi:10.1016/j.csbj.2023.05.003

Cited by 13 publications

(5 citation statements)

References 218 publications

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“…CFPS systems can be powerful platforms for research in biochemistry 1 , 2 nanotechnology 3 , biophysics 4 , synthetic biology 5 , 6 , and biomedicine 7 because they can synthesize diverse proteins in vitro, including enzymes 8 , membrane proteins 9 – 11 , and cytotoxic proteins 12 . CFPS systems can be encapsulated into lipid vesicles and artificial cell models can be constructed using a bottom-up approach using such lipid vesicles to facilitate functions and interactions of proteins synthesized using these CFPS systems into the lipid vesicles 13 – 18 .…”

Section: Introductionmentioning

confidence: 99%

Efficiency of transcription and translation of cell-free protein synthesis systems in cell-sized lipid vesicles with changing lipid composition determined by fluorescence measurements

Miwa,

Wakamori,

Ariyoshi

et al. 2024

Sci Rep

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To develop artificial cell models that mimic living cells, cell-sized lipid vesicles encapsulating cell-free protein synthesis (CFPS) systems are useful for protein expressions or artificial gene circuits for vesicle–vesicle communications. Therefore, investigating the transcriptional and translational properties of CFPS systems in lipid vesicles is important for maximizing the synthesis and functions of proteins. Although transcription and translation using CFPS systems inside lipid vesicles are more important than that outside lipid vesicles, the former processes are not investigated by changing the lipid composition of lipid vesicles. Herein, we investigated changes in transcription and translation using CFPS systems inside giant lipid vesicles (approximately 5–20 μm in diameter) caused by changing the lipid composition of lipid vesicles containing neutral, positively, and negatively charged lipids. After incubating for 30 min, 1 h, 2 h, and 4 h, the transcriptional and translational activities in these lipid vesicles were determined by detecting the fluorescence intensities of the fluorogenic RNA aptamer on the 3′-untranslated region of mRNA (transcription) and the fluorescent protein sfCherry (translation), respectively. The results revealed that transcriptional and translational activities in a lipid vesicle containing positively charged lipids were high when the protein was synthesized using the CFPS system inside the lipid vesicle. Thus, the present study provides an experimental basis for constructing complex artificial cell models using bottom-up approaches.

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Section: Introductionmentioning

confidence: 99%

Efficiency of transcription and translation of cell-free protein synthesis systems in cell-sized lipid vesicles with changing lipid composition determined by fluorescence measurements

Miwa,

Wakamori,

Ariyoshi

et al. 2024

Sci Rep

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“…Historically, cell-free protein expression (CFPE) systems have been based on E. coli lysate and used where the target protein would be toxic to the cellular systems traditionally used to express it [4,5]. However, these methods were typically associated with expensive methodology, reducing their utility in large-scale reactions; poor specific yields and an inability to recreate post-translational modifications (PTMs) present further difficulties [6]. Attempts to overcome these limitations have begun to show progress within the last decade.…”

Section: Introductionmentioning

confidence: 99%

“…Attempts to overcome these limitations have begun to show progress within the last decade. Eukaryotic lysates can include machinery capable of PTMs and yields have been increased thanks to the adoption of T7-polymerase systems to allow coupled transcription-translation within the same system [6]. Notably, a CFPE system based on Nicotiana tabacum BY-2 lysate is capable of PTM incorporation, high specific yields, and can be effectively scaled between the microlitre and litre ranges [7,8].…”

Section: Introductionmentioning

confidence: 99%

Cell-free expression and SMA copolymer encapsulation of a functional receptor tyrosine kinase disease variant, FGFR3-TACC3

Snow,

Wijesiriwardena,

Lane

et al. 2024

Preprint

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Despite their high clinical relevance, obtaining structural and biophysical data on transmembrane proteins has been bottlenecked by challenges involved in their expression. The inherent enzymatic activity of receptor tyrosine kinases (RTK) presents an additional hurdle to producing functional protein. The oncogenic fusion of proteins to such RTKs creates a particularly difficult-to-express protein subtype due to their high flexibility, lack of stability, and propensity for aggregation. One such protein is the fibroblast growth factor receptor 3 fused with transforming acidic coiled-coil-containing protein 3 (FGFR3-TACC3), which has failed to express to sufficient quality or functionality in traditional expression systems. Cell-free protein expression (CFPE) is a burgeoning arm of synthetic biology, enabling the rapid and efficient generation of recombinant proteins. This platform is characterised by utilising an optimised solution of cellular machinery to facilitate protein synthesisin vitro. In doing so, CFPE can act as a surrogate system for a range of proteins that are otherwise difficult to express through traditional host cell-based approaches. Here, functional FGFR3-TACC3 was expressed through a novel cell-free expression system in under 48 hours. The resultant protein can be reconstituted using SMA copolymers. Functionally, the protein demonstrated significant kinase domain phosphorylation (t<0.0001). Currently, there is no published, high-resolution structure of any full-length RTK. These findings form a promising foundation for future research on oncogenic RTKs and the application of cell-free systems for synthesising functional membrane proteins.

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“…Cell-free synthesis systems emerge as a promising solution in this scenario [14][15][16][17]. They enable the direct expression of genes from linear DNA without living cells, offering a flexible and controllable platform for swiftly developing enzymatic pathways.…”

Section: Introductionmentioning

confidence: 99%

Cell-Free Synthesis: Expediting Biomanufacturing of Chemical and Biological Molecules

Lee,

Kim

2024

Molecules

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The increasing demand for sustainable alternatives underscores the critical need for a shift away from traditional hydrocarbon-dependent processes. In this landscape, biomanufacturing emerges as a compelling solution, offering a pathway to produce essential chemical materials with significantly reduced environmental impacts. By utilizing engineered microorganisms and biomass as raw materials, biomanufacturing seeks to achieve a carbon-neutral footprint, effectively counteracting the carbon dioxide emissions associated with fossil fuel use. The efficiency and specificity of biocatalysts further contribute to lowering energy consumption and enhancing the sustainability of the production process. Within this context, cell-free synthesis emerges as a promising approach to accelerate the shift towards biomanufacturing. Operating with cellular machinery in a controlled environment, cell-free synthesis offers multiple advantages: it enables the rapid evaluation of biosynthetic pathways and optimization of the conditions for the synthesis of specific chemicals. It also holds potential as an on-demand platform for the production of personalized and specialized products. This review explores recent progress in cell-free synthesis, highlighting its potential to expedite the transformation of chemical processes into more sustainable biomanufacturing practices. We discuss how cell-free techniques not only accelerate the development of new bioproducts but also broaden the horizons for sustainable chemical production. Additionally, we address the challenges of scaling these technologies for commercial use and ensuring their affordability, which are critical for cell-free systems to meet the future demands of industries and fully realize their potential.

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Advancing synthetic biology through cell-free protein synthesis

Cited by 13 publications

References 218 publications

Efficiency of transcription and translation of cell-free protein synthesis systems in cell-sized lipid vesicles with changing lipid composition determined by fluorescence measurements

Efficiency of transcription and translation of cell-free protein synthesis systems in cell-sized lipid vesicles with changing lipid composition determined by fluorescence measurements

Cell-free expression and SMA copolymer encapsulation of a functional receptor tyrosine kinase disease variant, FGFR3-TACC3

Cell-Free Synthesis: Expediting Biomanufacturing of Chemical and Biological Molecules

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