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3 pages, 183 KiB

Open AccessEditorial

“Cell-Free Synthetic Biology”: Synthetic Biology Meets Cell-Free Protein Synthesis

by Seok Hoon Hong

Methods Protoc. 2019, 2(4), 80; https://doi.org/10.3390/mps2040080 - 8 Oct 2019

Cited by 2 | Viewed by 3279

Abstract

Since Nirenberg and Matthaei used cell-free protein synthesis (CFPS) to elucidate the genetic code in the early 1960s [...] Full article

(This article belongs to the Special Issue Cell-Free Synthetic Biology)

Research

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21 pages, 1756 KiB

Open AccessArticle

Accelerating the Production of Druggable Targets: Eukaryotic Cell-Free Systems Come into Focus

by Lena Thoring, Anne Zemella, Doreen Wüstenhagen and Stefan Kubick

Methods Protoc. 2019, 2(2), 30; https://doi.org/10.3390/mps2020030 - 16 Apr 2019

Cited by 14 | Viewed by 3948

Abstract

In the biopharmaceutical pipeline, protein expression systems are of high importance not only for the production of biotherapeutics but also for the discovery of novel drugs. The vast majority of drug targets are proteins, which need to be characterized and validated prior to [...] Read more.

In the biopharmaceutical pipeline, protein expression systems are of high importance not only for the production of biotherapeutics but also for the discovery of novel drugs. The vast majority of drug targets are proteins, which need to be characterized and validated prior to the screening of potential hit components and molecules. A broad range of protein expression systems is currently available, mostly based on cellular organisms of prokaryotic and eukaryotic origin. Prokaryotic cell-free systems are often the system of choice for drug target protein production due to the simple generation of expression hosts and low cost of preparation. Limitations in the production of complex mammalian proteins appear due to inefficient protein folding and posttranslational modifications. Alternative protein production systems, so-called eukaryotic cell-free protein synthesis systems based on eukaryotic cell-lysates, close the gap between a fast protein generation system and a high quality of complex mammalian proteins. In this study, we show the production of druggable target proteins in eukaryotic cell-free systems. Functional characterization studies demonstrate the bioactivity of the proteins and underline the potential for eukaryotic cell-free systems to significantly improve drug development pipelines. Full article

(This article belongs to the Special Issue Cell-Free Synthetic Biology)

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Review

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25 pages, 6167 KiB

Open AccessReview

Cell-Free Synthetic Biology Platform for Engineering Synthetic Biological Circuits and Systems

by Dohyun Jeong, Melissa Klocke, Siddharth Agarwal, Jeongwon Kim, Seungdo Choi, Elisa Franco and Jongmin Kim

Methods Protoc. 2019, 2(2), 39; https://doi.org/10.3390/mps2020039 - 14 May 2019

Cited by 22 | Viewed by 8430

Abstract

Synthetic biology integrates diverse engineering disciplines to create novel biological systems for biomedical and technological applications. The substantial growth of the synthetic biology field in the past decade is poised to transform biotechnology and medicine. To streamline design processes and facilitate debugging of [...] Read more.

Synthetic biology integrates diverse engineering disciplines to create novel biological systems for biomedical and technological applications. The substantial growth of the synthetic biology field in the past decade is poised to transform biotechnology and medicine. To streamline design processes and facilitate debugging of complex synthetic circuits, cell-free synthetic biology approaches has reached broad research communities both in academia and industry. By recapitulating gene expression systems in vitro, cell-free expression systems offer flexibility to explore beyond the confines of living cells and allow networking of synthetic and natural systems. Here, we review the capabilities of the current cell-free platforms, focusing on nucleic acid-based molecular programs and circuit construction. We survey the recent developments including cell-free transcription–translation platforms, DNA nanostructures and circuits, and novel classes of riboregulators. The links to mathematical models and the prospects of cell-free synthetic biology platforms will also be discussed. Full article

(This article belongs to the Special Issue Cell-Free Synthetic Biology)

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11 pages, 3138 KiB

Open AccessReview

Cell-Free Metabolic Engineering: Recent Developments and Future Prospects

by Hye Jin Lim and Dong-Myung Kim

Methods Protoc. 2019, 2(2), 33; https://doi.org/10.3390/mps2020033 - 30 Apr 2019

Cited by 25 | Viewed by 6168

Abstract

Due to the ongoing crises of fossil fuel depletion, climate change, and environmental pollution, microbial processes are increasingly considered as a potential alternative for cleaner and more efficient production of the diverse chemicals required for modern civilization. However, many issues, including low efficiency [...] Read more.

Due to the ongoing crises of fossil fuel depletion, climate change, and environmental pollution, microbial processes are increasingly considered as a potential alternative for cleaner and more efficient production of the diverse chemicals required for modern civilization. However, many issues, including low efficiency of raw material conversion and unintended release of genetically modified microorganisms into the environment, have limited the use of bioprocesses that rely on recombinant microorganisms. Cell-free metabolic engineering is emerging as a new approach that overcomes the limitations of existing cell-based systems. Instead of relying on metabolic processes carried out by living cells, cell-free metabolic engineering harnesses the metabolic activities of cell lysates in vitro. Such approaches offer several potential benefits, including operational simplicity, high conversion yield and productivity, and prevention of environmental release of microorganisms. In this article, we review the recent progress in this field and discuss the prospects of this technique as a next-generation bioconversion platform for the chemical industry. Full article

(This article belongs to the Special Issue Cell-Free Synthetic Biology)

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34 pages, 4464 KiB

Open AccessReview

A User’s Guide to Cell-Free Protein Synthesis

by Nicole E. Gregorio, Max Z. Levine and Javin P. Oza

Methods Protoc. 2019, 2(1), 24; https://doi.org/10.3390/mps2010024 - 12 Mar 2019

Cited by 150 | Viewed by 30292

Abstract

Cell-free protein synthesis (CFPS) is a platform technology that provides new opportunities for protein expression, metabolic engineering, therapeutic development, education, and more. The advantages of CFPS over in vivo protein expression include its open system, the elimination of reliance on living cells, and [...] Read more.

Cell-free protein synthesis (CFPS) is a platform technology that provides new opportunities for protein expression, metabolic engineering, therapeutic development, education, and more. The advantages of CFPS over in vivo protein expression include its open system, the elimination of reliance on living cells, and the ability to focus all system energy on production of the protein of interest. Over the last 60 years, the CFPS platform has grown and diversified greatly, and it continues to evolve today. Both new applications and new types of extracts based on a variety of organisms are current areas of development. However, new users interested in CFPS may find it challenging to implement a cell-free platform in their laboratory due to the technical and functional considerations involved in choosing and executing a platform that best suits their needs. Here we hope to reduce this barrier to implementing CFPS by clarifying the similarities and differences amongst cell-free platforms, highlighting the various applications that have been accomplished in each of them, and detailing the main methodological and instrumental requirement for their preparation. Additionally, this review will help to contextualize the landscape of work that has been done using CFPS and showcase the diversity of applications that it enables. Full article

(This article belongs to the Special Issue Cell-Free Synthetic Biology)

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Other

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15 pages, 2705 KiB

Open AccessProtocol

A Crude Extract Preparation and Optimization from a Genomically Engineered Escherichia coli for the Cell-Free Protein Synthesis System: Practical Laboratory Guideline

by Jeehye Kim, Caroline E. Copeland, Sahana R. Padumane and Yong-Chan Kwon

Methods Protoc. 2019, 2(3), 68; https://doi.org/10.3390/mps2030068 - 9 Aug 2019

Cited by 22 | Viewed by 8146

Abstract

With the advancement of synthetic biology, the cell-free protein synthesis (CFPS) system has been receiving the spotlight as a versatile toolkit for engineering natural and unnatural biological systems. The CFPS system reassembles the materials necessary for transcription and translation and recreates the in [...] Read more.

With the advancement of synthetic biology, the cell-free protein synthesis (CFPS) system has been receiving the spotlight as a versatile toolkit for engineering natural and unnatural biological systems. The CFPS system reassembles the materials necessary for transcription and translation and recreates the in vitro protein synthesis environment by escaping a physical living boundary. The cell extract plays an essential role in this in vitro format. Here, we propose a practical protocol and method for Escherichia coli-derived cell extract preparation and optimization, which can be easily applied to both commercially available and genomically engineered E. coli strains. The protocol includes: (1) The preparation step for cell growth and harvest, (2) the thorough step-by-step procedures for E. coli cell extract preparation including the cell wash and lysis, centrifugation, runoff reaction, and dialysis, (3) the preparation for the CFPS reaction components and, (4) the quantification of cell extract and cell-free synthesized protein. We anticipate that the protocol in this research will provide a simple preparation and optimization procedure of a highly active E. coli cell extract. Full article

(This article belongs to the Special Issue Cell-Free Synthetic Biology)

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8 pages, 901 KiB

Open AccessBenchmark

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

by Seung-Ook Yang, Gregory H. Nielsen, Kristen M. Wilding, Merideth A. Cooper, David W. Wood and Bradley C. Bundy

Methods Protoc. 2019, 2(2), 52; https://doi.org/10.3390/mps2020052 - 21 Jun 2019

Cited by 7 | Viewed by 4581

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue Cell-Free Synthetic Biology)

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12 pages, 994 KiB

Open AccessBenchmark

Optimizing Cell-Free Protein Synthesis for Increased Yield and Activity of Colicins

by Xing Jin, Weston Kightlinger and Seok Hoon Hong

Methods Protoc. 2019, 2(2), 28; https://doi.org/10.3390/mps2020028 - 11 Apr 2019

Cited by 18 | Viewed by 4506

Abstract

Colicins are antimicrobial proteins produced by Escherichia coli that hold great promise as viable complements or alternatives to antibiotics. Cell-free protein synthesis (CFPS) is a useful production platform for toxic proteins because it eliminates the need to maintain cell viability, a common problem [...] Read more.

Colicins are antimicrobial proteins produced by Escherichia coli that hold great promise as viable complements or alternatives to antibiotics. Cell-free protein synthesis (CFPS) is a useful production platform for toxic proteins because it eliminates the need to maintain cell viability, a common problem in cell-based production. Previously, we demonstrated that colicins produced by CFPS based on crude Escherichia coli lysates are effective in eradicating antibiotic-tolerant bacteria known as persisters. However, we also found that some colicins have poor solubility or low cell-killing activity. In this study, we improved the solubility of colicin M from 16% to nearly 100% by producing it in chaperone-enriched E. coli extracts, resulting in enhanced cell-killing activity. We also improved the cytotoxicity of colicin E3 by adding or co-expressing the E3 immunity protein during the CFPS reaction, suggesting that the E3 immunity protein enhances colicin E3 activity in addition to protecting the host strain. Finally, we confirmed our previous finding that active colicins can be rapidly synthesized by observing colicin E1 production over time in CFPS. Within three hours of CFPS incubation, colicin E1 reached its maximum production yield and maintained high cytotoxicity during longer incubations up to 20 h. Taken together, our findings indicate that colicin production can be easily optimized for improved solubility and activity using the CFPS platform. Full article

(This article belongs to the Special Issue Cell-Free Synthetic Biology)

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12 pages, 2425 KiB

Open AccessProtocol

Efficient Incorporation of Unnatural Amino Acids into Proteins with a Robust Cell-Free System

by Wei Gao, Ning Bu and Yuan Lu

Methods Protoc. 2019, 2(1), 16; https://doi.org/10.3390/mps2010016 - 12 Feb 2019

Cited by 18 | Viewed by 6364

Abstract

Unnatural proteins are crucial biomacromolecules and have been widely applied in fundamental science, novel biopolymer materials, enzymes, and therapeutics. Cell-free protein synthesis (CFPS) system can serve as a robust platform to synthesize unnatural proteins by highly effective site-specific incorporation of unnatural amino acids [...] Read more.

Unnatural proteins are crucial biomacromolecules and have been widely applied in fundamental science, novel biopolymer materials, enzymes, and therapeutics. Cell-free protein synthesis (CFPS) system can serve as a robust platform to synthesize unnatural proteins by highly effective site-specific incorporation of unnatural amino acids (UNAAs), without the limitations of cell membrane permeability and the toxicity of unnatural components. Here, we describe a quick and simple method to synthesize unnatural proteins in CFPS system based on Escherichia coli crude extract, with unnatural orthogonal aminoacyl-tRNA synthetase and suppressor tRNA evolved from Methanocaldococcus jannaschii. The superfolder green fluorescent protein (sfGFP) and p-propargyloxyphenylalanine (pPaF) were used as the model protein and UNAA. The synthesis of unnatural sfGFPs was characterized by microplate spectrophotometer, affinity chromatography, and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). This protocol provides a detailed procedure guiding how to use the powerful CFPS system to synthesize unnatural proteins on demand. Full article

(This article belongs to the Special Issue Cell-Free Synthetic Biology)

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