ChemInform Abstract: Cell‐Free Protein Synthesis: Pros and Cons of Prokaryotic and Eukaryotic Systems

Zemella, Anne; Thoring, Lena; Hoffmeister, Christian; Kubick, Stefan

doi:10.1002/chin.201603212

Cited by 8 publications

(9 citation statements)

References 139 publications

(187 reference statements)

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“…Comparing costs between prokaryotic lysate-based and eukaryotic lysate-based CFPS systems, it is demonstrated that despite the fact that prokaryotic CFPS systems are deficient in performing posttranslational modifications, they have proved to have higher yields at lower costs of extract preparation (Jin & Hong, 2018;Zemella et al, 2015). CFPS first generations suffered low protein expression yields and prolonged processing times due to nutrients/ energy depletion and waste-product accumulation, all of which have been resolved since the advent of continuous-flow formats (Katzen et al, 2005).…”

Section: Challenges and Future Directionsmentioning

confidence: 99%

Cell‐free protein synthesis: The transition from batch reactions to minimal cells and microfluidic devices

Ayoubi‐Joshaghani

Dianat‐Moghadam

Seidi

et al. 2020

Biotech & Bioengineering

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Thanks to the synthetic biology, the laborious and restrictive procedure for producing a target protein in living microorganisms by biotechnological approaches can now experience a robust, pliant yet efficient alternative. The new system combined with lab‐on‐chip microfluidic devices and nanotechnology offers a tremendous potential envisioning novel cell‐free formats such as DNA brushes, hydrogels, vesicular particles, droplets, as well as solid surfaces. Acting as robust microreactors/microcompartments/minimal cells, the new platforms can be tuned to perform various tasks in a parallel and integrated manner encompassing gene expression, protein synthesis, purification, detection, and finally enabling cell‐cell signaling to bring a collective cell behavior, such as directing differentiation process, characteristics of higher order entities, and beyond. In this review, we issue an update on recent cell‐free protein synthesis (CFPS) formats. Furthermore, the latest advances and applications of CFPS for synthetic biology and biotechnology are highlighted. In the end, contemporary challenges and future opportunities of CFPS systems are discussed.

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Section: Challenges and Future Directionsmentioning

confidence: 99%

Cell‐free protein synthesis: The transition from batch reactions to minimal cells and microfluidic devices

Ayoubi‐Joshaghani

Dianat‐Moghadam

Seidi

et al. 2020

Biotech & Bioengineering

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“…Extracts from prokaryotes (e.g., E. coli) and eukaryotes (e.g., wheat germ), along with rabbit reticulocytes and mammalian cell extracts have been widely used for protein synthesis in vitro (Bernhard & Tozawa, 2013;Endo & Sawasaki, 2006;Zemella, Thoring, Hoffmeister, & Kubick, 2015).…”

Section: A R Ia N T S Of Th E C a N O Ni C A L Eg G E X T R A C Tmentioning

confidence: 99%

Probing the biology of cell boundary conditions through confinement of Xenopus cell‐free cytoplasmic extracts

Bermudez

Chen

Einstein

et al. 2017

Genesis

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Cell-free cytoplasmic extracts prepared from Xenopus eggs and embryos have for decades provided a biochemical system with which to interrogate complex cell biological processes in vitro. Recently, the application of microfabrication and microfluidic strategies in biology has narrowed the gap between in vitro and in vivo studies by enabling formation of cell-size compartments containing functional cytoplasm. These approaches provide numerous advantages over traditional biochemical experiments performed in a test tube. Most notably, the cell-free cytoplasm is confined using a two- or three-dimensional boundary, which mimics the natural configuration of a cell. This strategy enables characterization of the spatial organization of a cell, and the role that boundaries play in regulating intracellular assembly and function. In this review we describe the marriage of Xenopus cell-free cytoplasm and confinement technologies to generate synthetic cell-like systems, the recent biological insights they have enabled, and the promise they hold for future scientific discovery.

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“…Cell-free protein synthesis (CFPS) has gained much attention as an emerging technology in synthetic biology with a number of recent accounts in the literature (Chen and Jewett, 2016;Kahlig, 2014;Smith et al, 2014;Zemella et al, 2015). Among the many attractive attributes of CFPS as an alternative to cell-based protein expression methods (Swartz, 2012), is its demonstrated potential in possibly addressing the current need for on-demand, point-of-care production of protein biologics (Mohr et al, 2016;Pardee et al, 2016;Sullivan et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

“…A comprehensive review (Zemella et al, 2015) compares various CFPS systems, which includes a detailed discussion of the advantages of using mammalian-based Chinese Hamster Ovary (CHO) cell extracts for the production of protein biologics over other well-known systems. For complex therapeutic proteins requiring post-translational modifications, a CHO-based CFPS system is an ideal platform for efficient, high-throughput synthesis.…”

Section: Introductionmentioning

confidence: 99%

Optimizing cell‐free protein expression in CHO: Assessing small molecule mass transfer effects in various reactor configurations

Peñalber-Johnstone

Andar

Tran

et al. 2017

Biotech & Bioengineering

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Cell-free protein synthesis (CFPS) is an ideal platform for rapid and convenient protein production. However, bioreactor design remains a critical consideration in optimizing protein expression. Using turbo green fluorescent protein (tGFP) as a model, we tracked small molecule components in a Chinese Hamster Ovary (CHO) CFPS system to optimize protein production. Here, three bioreactors in continuous-exchange cell-free (CECF) format were characterized. A GFP optical sensor was built to monitor the product in real-time. Mass transfer of important substrate and by-product components such as nucleoside triphosphates (NTPs), creatine, and inorganic phosphate (Pi) across a 10-kDa MWCO cellulose membrane was calculated. The highest efficiency measured by tGFP yields were found in a microdialysis device configuration; while a negative effect on yield was observed due to limited mass transfer of NTPs in a dialysis cup configuration. In 24-well plate high-throughput CECF format, addition of up to 40 mM creatine phosphate in the system increased yields by up to ∼60% relative to controls. Direct ATP addition, as opposed to creatine phosphate addition, negatively affected the expression. Pi addition of up to 30 mM to the expression significantly reduced yields by over ∼40% relative to controls. Overall, data presented in this report serves as a valuable reference to optimize the CHO CFPS system for next-generation bioprocessing. Biotechnol. Bioeng. 2017;114: 1478-1486. © 2017 Wiley Periodicals, Inc.

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ChemInform Abstract: Cell‐Free Protein Synthesis: Pros and Cons of Prokaryotic and Eukaryotic Systems

Abstract: Review: 177 refs.

Cited by 8 publications

References 139 publications

Cell‐free protein synthesis: The transition from batch reactions to minimal cells and microfluidic devices

Cell‐free protein synthesis: The transition from batch reactions to minimal cells and microfluidic devices

Probing the biology of cell boundary conditions through confinement of Xenopus cell‐free cytoplasmic extracts

Optimizing cell‐free protein expression in CHO: Assessing small molecule mass transfer effects in various reactor configurations

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