Energizing eukaryotic cell‐free protein synthesis with glucose metabolism

Anderson, Mark J.; Stark, Jessica C.; Hodgman, C. Eric; Jewett, Michael C.

doi:10.1016/j.febslet.2015.05.045

Cited by 32 publications

(44 citation statements)

References 43 publications

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“…At least the costs of exogenous added T7‐RNA‐polymerase might be decreased by in‐house‐production. The development of alternative energy‐rich components and the energy regeneration systems is already in process (Anderson et al, ). In summary, the increasing productivity of eukaryotic cell‐free systems, in combination with the reduced costs for lysates and the energy regeneration system are excellent preconditions to increase the yield‐on‐cost ratio significantly.…”

Section: Discussionmentioning

confidence: 99%

Production of G protein‐coupled receptors in an insect‐based cell‐free system

Sonnabend

Spahn

Stech

et al. 2017

Biotech & Bioengineering

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The biochemical analysis of human cell membrane proteins remains a challenging task due to the difficulties in producing sufficient quantities of functional protein. G protein‐coupled receptors (GPCRs) represent a main class of membrane proteins and drug targets, which are responsible for a huge number of signaling processes regulating various physiological functions in living cells. To circumvent the current bottlenecks in GPCR studies, we propose the synthesis of GPCRs in eukaryotic cell‐free systems based on extracts generated from insect (Sf21) cells. Insect cell lysates harbor the fully active translational and translocational machinery allowing posttranslational modifications, such as glycosylation and phosphorylation of de novo synthesized proteins. Here, we demonstrate the production of several GPCRs in a eukaryotic cell‐free system, performed within a short time and in a cost‐effective manner. We were able to synthesize a variety of GPCRs ranging from 40 to 133 kDa in an insect‐based cell‐free system. Moreover, we have chosen the μ opioid receptor (MOR) as a model protein to analyze the ligand binding affinities of cell‐free synthesized MOR in comparison to MOR expressed in a human cell line by “one‐point” radioligand binding experiments. Biotechnol. Bioeng. 2017;114: 2328–2338. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

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Section: Discussionmentioning

confidence: 99%

Production of G protein‐coupled receptors in an insect‐based cell‐free system

Sonnabend

Spahn

Stech

et al. 2017

Biotech & Bioengineering

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“…Huge efforts have been invested to introduce a novel energy regeneration system into yeast-based CFPS. 76 As for the E. coli system, the rapid production of phosphates from high-energy compounds such as phosphoenolpyruvate, creatine phosphate, and acetyl phosphate has an inhibitory effect on CFPS. Moreover, these high-energy compounds are expensive and resulted in limited industrial application.…”

Section: Fungal Extractsmentioning

confidence: 99%

“… 27 Studies have proven that the use of glucose in combination with phosphate, as alternative energy source, results in more-efficient ATP production in comparison to standard energy sources such as creatine phosphate and phosphoenolpyruvate. 76 Moreover, the accumulation of inhibitory phosphates was avoided. By using this novel energy system, protein yields of up to 3.64 μg mL −1 active luciferase were obtained.…”

Section: Fungal Extractsmentioning

confidence: 99%

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

et al. 2015

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From its start as a small‐scale in vitro system to study fundamental translation processes, cell‐free protein synthesis quickly rose to become a potent platform for the high‐yield production of proteins. In contrast to classical in vivo protein expression, cell‐free systems do not need time‐consuming cloning steps, and the open nature provides easy manipulation of reaction conditions as well as high‐throughput potential. Especially for the synthesis of difficult to express proteins, such as toxic and transmembrane proteins, cell‐free systems are of enormous interest. The modification of the genetic code to incorporate non‐canonical amino acids into the target protein in particular provides enormous potential in biotechnology and pharmaceutical research and is in the focus of many cell‐free projects. Many sophisticated cell‐free systems for manifold applications have been established. This review describes the recent advances in cell‐free protein synthesis and details the expanding applications in this field.

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“…CFPS has been developed with several different organisms (e.g. E. coli , wheat germ, rabbit reticulocytes, CHO cells, yeast, and insect cells) to produce various proteins including therapeutic vaccines, virus‐like particles, and membrane proteins . CFPS is also being used for high‐throughput functional and structural studies of a wide variety of proteins , as well as more recently for rapid prototyping of biological circuits .…”

Section: Introductionmentioning

confidence: 99%

Cell‐free protein synthesis enables high yielding synthesis of an active multicopper oxidase

Lawton

Kostecki

et al. 2015

Biotechnology Journal

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Multicopper oxidases (MCOs) are broadly distributed in all kingdoms of life and perform a variety of important oxidative reactions. These enzymes have potential biotechnological applications; however, the applications are impeded by low expression yields in traditional recombinant hosts, solubility issues, and poor copper cofactor assembly. As an alternative to traditional recombinant protein expression, we show the ability to use cell‐free protein synthesis (CFPS) to produce complex MCO proteins with high soluble titers. Specifically, we report the production of MCOs in an Escherichia coli‐based cell‐free transcription‐translation system. Total yields as high as 1.2 mg mL−1 were observed after a 20‐h batch reaction. More than 95% of the protein was soluble and activity was obtained by simple post‐CFPS addition of copper ions in the form of CuSO4. Scale‐up reactions were achieved from 15 to 100 µL without a decrease in productivity and solubility. CFPS titers were higher than in vivo expression titers and more soluble, avoiding the formation of inclusion bodies. Our work extends the utility of the cell‐free platform to the production of active proteins containing copper cofactors and demonstrates a simple method for producing MCOs.

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Energizing eukaryotic cell‐free protein synthesis with glucose metabolism

Cited by 32 publications

References 43 publications

Production of G protein‐coupled receptors in an insect‐based cell‐free system

Production of G protein‐coupled receptors in an insect‐based cell‐free system

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

Cell‐free protein synthesis enables high yielding synthesis of an active multicopper oxidase

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