Creating a completely “cell‐free” system for protein synthesis

Smith, Mark T.; Bennett, Anthony M.; Hunt, Jeremy M.; Bundy, Bradley C.

doi:10.1002/btpr.2157

Cited by 33 publications

(22 citation statements)

References 20 publications

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“…This issue will need to be carefully addressed should the sensors be used outside of the laboratory setting. To this end, adjusting lyophilization conditions (40), increasing the intracellular lysis protein levels, and adding bactericidal agents that do not interfere with cell-free expression could feasibly be employed, however a careful further investigation of the issue is required.…”

Section: Discussionmentioning

confidence: 99%

Rapid and Scalable Preparation of Bacterial Lysates for Cell-Free Gene Expression

et al. 2017

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Cell-free gene expression systems are emerging as an important platform for a diverse range of synthetic biology and biotechnology applications, including production of robust field-ready biosensors. Here, we combine programmed cellular autolysis with a freeze-thaw or freeze-dry cycle to create a practical, reproducible, and a labor- and cost-effective approach for rapid production of bacterial lysates for cell-free gene expression. Using this method, robust and highly active bacterial cell lysates can be produced without specialized equipment at a wide range of scales, making cell-free gene expression easily and broadly accessible. Moreover, live autolysis strain can be freeze-dried directly and subsequently lysed upon rehydration to produce active lysate. We demonstrate the utility of autolysates for synthetic biology by regulating protein production and degradation, implementing quorum sensing, and showing quantitative protection of linear DNA templates by GamS protein. To allow versatile and sensitive β-galactosidase (LacZ) based readout we produce autolysates with no detectable background LacZ activity and use them to produce sensitive mercury(II) biosensors with LacZ-mediated colorimetric and fluorescent outputs. The autolysis approach can facilitate wider adoption of cell-free technology for cell-free gene expression as well as other synthetic biology and biotechnology applications, such as metabolic engineering, natural product biosynthesis, or proteomics.

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Section: Discussionmentioning

confidence: 99%

Rapid and Scalable Preparation of Bacterial Lysates for Cell-Free Gene Expression

et al. 2017

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“…Cell extracts were made using either E. coli BL21 Star (DE3) (Invitrogen, Carlsbad, CA) or ClearColi BL21 DE3 (Lucigen, Middleton, WI). Extract was produced as described previously with either LB, 2xYT, or TB fermentation media …”

Section: Methodsmentioning

confidence: 99%

Endotoxin-Free E. coli- Based Cell-Free Protein Synthesis: Pre-Expression Endotoxin Removal Approaches for on-Demand Cancer Therapeutic Production

et al. 2018

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Approximately one third of protein therapeutics are produced in Escherichia coli, targeting a wide variety of diseases. However, due to immune recognition of endotoxin (a lipid component in the E. coli cell membrane), these protein products must be extensively purified before application to avoid adverse reactions such as septic shock. E. coli-based cell-free protein synthesis (CFPS), which has emerged as a promising platform for the development and production of enhanced protein therapeutics, provides a unique opportunity to remove endotoxins prior to protein expression due to its open environment and the absence of live cells. Pre-expression endotoxin removal from CFPS reagents could simplify downstream processing, potentially enabling on-demand production of unique protein therapeutics. Herein, three strategies for removing endotoxins from E. coli cell lysate are evaluated: Triton X-114 two-phase extraction, polylysine affinity chromatography, and extract preparation from genetically engineered, endotoxin-free ClearColi cells. It is demonstrated that current protocols for endotoxin removal treatments insufficiently reduce endotoxin and significantly reduce protein synthesis yields. Further, the first adaptation of ClearColi cells to prepare cell-free extract with high protein synthesis capability is demonstrated. Finally, production of the acute lymphoblastic leukemia therapeutic crisantaspase from reduced-endotoxin extract and endotoxin-free ClearColi extract is demonstrated.

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“…Our lab demonstrated that we can efficiently produce a cytotoxic anticancer therapeutic, onconase, with a year old lyophilized cell extract (Salehi et al, 2016). Also, to create a portable CFPS platform to produce biotherapeutics and specially personalized/portable medicine and diagnostics, we reported how lyophilization can be used to sterilize and decontaminate cell extract without losing protein production capability (Smith et al, 2015). Additionally, a group of researchers recently reported a lyophilized, paper-based, in vitro system for synthetic gene network, which can expend the applications of the system into different areas including the clinic and global health (Pardee et al, 2014).…”

Section: Lyophilized Cell Extractmentioning

confidence: 99%

Escherichia coli-based cell-free extract development for protein-based cancer therapeutic production

Salehi¹,

Earl²,

Muhlestein³

et al. 2016

Int. J. Dev. Biol.

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Cell-free protein synthesis has been around for decades but it has never been close to becoming a robust tool for the production of biotherapeutic agents. In this review, we focus on how Escherichia coli-based cell-free protein synthesis can be modified in various ways to produce challenging, complex anticancer biotherapeutics. Here we report progress in extract preparation and its relation to cell-free cancer research. The future prospects of cell-free technology and its potential in various areas of cancer therapeutics production are also highlighted.

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Creating a completely “cell‐free” system for protein synthesis

Cited by 33 publications

References 20 publications

Rapid and Scalable Preparation of Bacterial Lysates for Cell-Free Gene Expression

Rapid and Scalable Preparation of Bacterial Lysates for Cell-Free Gene Expression

Endotoxin-Free E. coli- Based Cell-Free Protein Synthesis: Pre-Expression Endotoxin Removal Approaches for on-Demand Cancer Therapeutic Production

Escherichia coli-based cell-free extract development for protein-based cancer therapeutic production

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