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

Salehi, Amin S. M.; Earl, Conner C.; Muhlestein, Christina; Bundy, Bradley C.

doi:10.1387/ijdb.160125bb

Cited by 10 publications

(5 citation statements)

References 67 publications

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“…An engineered reporter enzyme domain on the biosensor protein exhibits ligand-dependent activity, resulting in a simple, colorimetric readout. Unique CFPS characteristics, including its chemically accessible reaction environment, robustness, scalability, and control, − make this technology a powerful biosensing platform for both simple and complex detection applications. In addition, the ability to lyophilize the CFPS components enables this type of biosensor to be stockpiled for emergencies and biothreat situations.…”

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confidence: 99%

Cell-Free Protein Synthesis Approach to Biosensing hTRβ-Specific Endocrine Disruptors

et al. 2017

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Here we introduce a Rapid Adaptable Portable In vitro Detection biosensor platform (RAPID) for detecting ligands that interact with nuclear hormone receptors (NHRs). The RAPID platform can be adapted for field use, allowing rapid evaluation of endocrine disrupting chemicals (EDCs) presence or absence in environmental samples, and can also be applied for drug screening. The biosensor is based on an engineered, allosterically activated fusion protein, which contains the ligand binding domain from a target NHR (human thyroid receptor β in this work). In vitro expression of this protein using cell-free protein synthesis (CFPS) technology in the presence of an EDC leads to activation of a reporter enzyme, reported through a straightforward colorimetric assay output. In this work, we demonstrate the potential of this biosensor platform to be used in a portable "just-add-sample" format for near real-time detection. We also demonstrate the robust nature of the cell-free protein synthesis component in the presence of a variety of environmental and human samples, including sewage, blood, and urine. The presented RAPID biosensor platform is significantly faster and less labor intensive than commonly available technologies, making it a promising tool for detecting environmental EDC contamination and screening potential NHR-targeted pharmaceuticals.

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confidence: 99%

Cell-Free Protein Synthesis Approach to Biosensing hTRβ-Specific Endocrine Disruptors

et al. 2017

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“…Hence, CFPS (recently also called TXTL for "transcription-translation") is getting more attention these days with the development of new methods that try to make the best use of the unique features of an in vitro method rather than relying on established systems depending on a host cell (Zemella et al, 2015) for synthesis of recombinant proteins. The high potential of new CFPS systems was demonstrated by an E. coli system that is used for protein expression on an industrial scale (Zawada et al, 2011;Salehi et al, 2016;Hershewe et al, 2020). It was suggested that such systems could be used more in the future for the production of dedicated pharma proteins, for example, incorporating noncanonical amino acids (Hong et al, 2014;Quast et al, 2015;Wu et al, 2020), preparation of dedicated proteins that were produced under more defined conditions than possible in cellbased systems (Oza et al, 2015), or allowing for the "on-demand" production of protein therapeutics in the clinic (Mohr et al, 2016;Sullivan et al, 2016;Timm et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

Easy Synthesis of Complex Biomolecular Assemblies: Wheat Germ Cell-Free Protein Expression in Structural Biology

Fogeron

Lecoq

Cole

et al. 2021

Front. Mol. Biosci.

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Cell-free protein synthesis (CFPS) systems are gaining more importance as universal tools for basic research, applied sciences, and product development with new technologies emerging for their application. Huge progress was made in the field of synthetic biology using CFPS to develop new proteins for technical applications and therapy. Out of the available CFPS systems, wheat germ cell-free protein synthesis (WG-CFPS) merges the highest yields with the use of a eukaryotic ribosome, making it an excellent approach for the synthesis of complex eukaryotic proteins including, for example, protein complexes and membrane proteins. Separating the translation reaction from other cellular processes, CFPS offers a flexible means to adapt translation reactions to protein needs. There is a large demand for such potent, easy-to-use, rapid protein expression systems, which are optimally serving protein requirements to drive biochemical and structural biology research. We summarize here a general workflow for a wheat germ system providing examples from the literature, as well as applications used for our own studies in structural biology. With this review, we want to highlight the tremendous potential of the rapidly evolving and highly versatile CFPS systems, making them more widely used as common tools to recombinantly prepare particularly challenging recombinant eukaryotic proteins.

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“…Pharmaceutical research and development over the past few decades has discovered treatments for a multitude of diseases including stroke [1], cancer [2] and heart disease [3]. Although these innovative treatments have provided much relief, there is still an economic barrier for many patients in need due to the high cost of drug treatments.…”

Section: Introductionmentioning

confidence: 99%

Towards On-Demand E. coli-Based Cell-Free Protein Synthesis of Tissue Plasminogen Activator

Yang

Nielsen

Wilding

et al. 2019

MPs

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Stroke is the leading cause of death with over 5 million deaths worldwide each year. About 80% of strokes are ischemic strokes caused by blood clots. Tissue plasminogen activator (tPa) is the only FDA-approved drug to treat ischemic stroke with a wholesale price over $6000. tPa is now off patent although no biosimilar has been developed. The production of tPa is complicated by the 17 disulfide bonds that exist in correctly folded tPA. Here, we present an Escherichia coli-based cell-free protein synthesis platform for tPa expression and report conditions which resulted in the production of active tPa. While the activity is below that of commercially available tPa, this work demonstrates the potential of cell-free expression systems toward the production of future biosimilars. The E. coli-based cell-free system is increasingly becoming an attractive platform for low-cost biosimilar production due to recent developments which enable production from shelf-stable lyophilized reagents, the removal of endotoxins from the reagents to prevent the risk of endotoxic shock, and rapid on-demand production in hours.

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

Cited by 10 publications

References 67 publications

Cell-Free Protein Synthesis Approach to Biosensing hTRβ-Specific Endocrine Disruptors

Cell-Free Protein Synthesis Approach to Biosensing hTRβ-Specific Endocrine Disruptors

Easy Synthesis of Complex Biomolecular Assemblies: Wheat Germ Cell-Free Protein Expression in Structural Biology

Towards On-Demand E. coli-Based Cell-Free Protein Synthesis of Tissue Plasminogen Activator

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