The growing impact of lyophilized cell-free protein expression systems

Hunt, J. Porter; Yang, Seung Ook; Wilding, Kristen M.; Bundy, Bradley C.

doi:10.1080/21655979.2016.1241925

Cited by 44 publications

(32 citation statements)

References 44 publications

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“…There has recently been a surge of interest in ‘in vitro biology’ [ 38 – 40 ], with in vitro transcription and translation (IVTT) reactions being used for diagnostic applications [ 41 ] and for the production of many new proteins [ 42 – 45 ]. Simplification of the traditionally labor and cost intensive procedures for preparation and storage of E .…”

Section: Discussionmentioning

confidence: 99%

Cellular reagents for diagnostics and synthetic biology

et al. 2018

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We have found that the overproduction of enzymes in bacteria followed by their lyophilization leads to 'cellular reagents' that can be directly used to carry out numerous molecular biology reactions. We demonstrate the use of cellular reagents in a variety of molecular diagnostics, such as TaqMan qPCR with no diminution in sensitivity, and in synthetic biology cornerstones such as the Gibson assembly of DNA fragments, where new plasmids can be constructed solely based on adding cellular reagents. Cellular reagents have significantly reduced complexity and cost of production, storage and implementation, features that should facilitate accessibility and use in resource-poor conditions.

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Section: Discussionmentioning

confidence: 99%

Cellular reagents for diagnostics and synthetic biology

et al. 2018

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“…The activity of cell-free-derived sfGFP was determined using an in-extract fluorescence analysis as described previously (39). Briefly, 2 μL of cell-free reaction product was diluted into 48 μL of Ambion nanopure water (Invitrogen, USA).…”

Section: Gfp Fluorescence Assaymentioning

confidence: 99%

“…A body of work dedicated to optimization of extract preparation and reaction conditions has simplified, expedited, and improved the cost and performance of E. coli CFE systems (22,28,29). Optimized E. coli-based CFE reactions: (i) quickly synthesize grams of protein per liter in batch reactions (30)(31)(32), (ii) are scalable from the nL to 100 L scale (33,34), and (iii) can be freeze-dried for months of shelf-stability and distribution to the point of care (6,22,29,(35)(36)(37)(38)(39)(40). Freeze-dried CFE systems are poised to make disruptive impacts in biotechnology, having already been leveraged for point-of-use biosensing (41)(42)(43)(44)(45)(46), therapeutic and vaccine production (37,38,47), and educational kits (22,(48)(49)(50).…”

Section: Main Text Introductionmentioning

confidence: 99%

Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles

Hershewe

Warfel

Iyer

et al. 2020

Preprint

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Cell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for accelerating the design of cellular function, on-demand biomanufacturing, portable diagnostics, and educational kits. Many essential biological processes that could endow CFE systems with desired functions, such as protein glycosylation, rely on the activity of membrane-bound components. However, without the use of synthetic membrane mimics, activating membrane-dependent functionality in bacterial CFE systems remains largely unstudied. Here, we address this gap by characterizing native, cell-derived membrane vesicles in Escherichia coli-based CFE extracts and describing methods to enrich vesicles with heterologous, membrane-bound machinery. We first use nanocharacterization techniques to show that lipid vesicles in CFE extracts are tens to hundreds of nanometers across, and on the order of ~3x1012 particles/mL. We then determine how extract processing methods, such as post-lysis centrifugation, can be used to modulate concentrations of membrane vesicles in CFE systems. By tuning these methods, we show that increasing the number of vesicle particles to ~7x1012 particles/mL can be used to increase concentrations of heterologous membrane protein cargo expressed prior to lysis. Finally, we apply our methods to enrich membrane-bound oligosaccharyltransferases and lipid-linked oligosaccharides for improving N-linked and O-linked glycoprotein synthesis. We anticipate that our findings will facilitate in vitro gene expression systems that require membrane-dependent activities and open new opportunities in glycoengineering.

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“…Cell‐free protein synthesis (CFPS) offers an alternative platform for ASNase activity assessment which presents multiple advantages over traditional approaches. First, CFPS reactions can be rendered shelf stable, thus mitigating the need for cold‐chain storage of assay components, enabling high‐density storage and facile use . Second, the CFPS platform provides an open reaction environment which may be dynamically optimized and is reportedly robust against samples containing human blood .…”

Section: Introductionmentioning

confidence: 99%

Engineering Cell‐Free Protein Synthesis for High‐Yield Production and Human Serum Activity Assessment of Asparaginase: Toward On‐Demand Treatment of Acute Lymphoblastic Leukemia

Hunt

Wilding

Barnett

et al. 2020

Biotechnology Journal

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Acute lymphocytic leukemia (ALL) is a common childhood cancer in the United States, with over 6000 new cases diagnosed each year. Administration of bacterial asparaginase (ASNase) has improved survival rates to nearly 80%, however these therapeutics have high incidence of immunological neutralization and serum activity must be monitored for most effective treatment regimens. Here, a 72% improvement in cell‐free protein synthesis (CFPS) of FDA approved l‐asparaginase (crisantaspase) is demonstrated by employing an aspartate‐fed‐batch reactor format. A CFPS‐based ASNase activity assay as a tool for therapeutic regimentation and production quality control is also presented. This work suggests that shelf‐stable and low‐cost Escherichia coli‐based CFPS reactions may be employed on‐demand to 1) synthesize biologics on‐site for patient administration, 2) verify biologic activity for dosage calculations, and 3) monitor therapeutic activity in human serum during the treatment regimen. The combination of both therapeutic production and activity assessment introduces a concept of synergistic utility for bacterial cell lysates in modern medical treatment. Indeed, recent work with CFPS biosensors supports a not‐too‐distant future when shelf‐stable E. coli CFPS systems are used to diagnose, treat, and monitor treatment of diseases in the clinical setting.

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The growing impact of lyophilized cell-free protein expression systems

Cited by 44 publications

References 44 publications

Cellular reagents for diagnostics and synthetic biology

Cellular reagents for diagnostics and synthetic biology

Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles

Engineering Cell‐Free Protein Synthesis for High‐Yield Production and Human Serum Activity Assessment of Asparaginase: Toward On‐Demand Treatment of Acute Lymphoblastic Leukemia

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