Cell-free protein synthesis: Recent advances in bacterial extract sources and expanded applications

Liu, Wanqiu; Zhang, Lingkai; Chen, Mingzhe; Li, Jian

doi:10.1016/j.bej.2018.10.023

Cited by 57 publications

(41 citation statements)

References 103 publications

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“…Both approaches contain all necessary constituents for the coupled transcription and translation machinery, such as ribosomes, aminoacyl-tRNA-synthetases, and translation factors for initiation, elongation, and product release ( Figure 3). Various crude cell extracts obtained from prokaryotic, fungi, plant, and mammalian cells are described in literature [41,42]. In general, any organism can serve as the source for cell extract preparation.…”

Section: Cell-free Protein Synthesismentioning

confidence: 99%

Application of Cell-Free Protein Synthesis for Faster Biocatalyst Development

2019

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Cell-free protein synthesis (CFPS) has become an established tool for rapid protein synthesis in order to accelerate the discovery of new enzymes and the development of proteins with improved characteristics. Over the past years, progress in CFPS system preparation has been made towards simplification, and many applications have been developed with regard to tailor-made solutions for specific purposes. In this review, various preparation methods of CFPS systems are compared and the significance of individual supplements is assessed. The recent applications of CFPS are summarized and the potential for biocatalyst development discussed. One of the central features is the high-throughput synthesis of protein variants, which enables sophisticated approaches for rapid prototyping of enzymes. These applications demonstrate the contribution of CFPS to enhance enzyme functionalities and the complementation to in vivo protein synthesis. However, there are different issues to be addressed, such as the low predictability of CFPS performance and transferability to in vivo protein synthesis. Nevertheless, the usage of CFPS for high-throughput enzyme screening has been proven to be an efficient method to discover novel biocatalysts and improved enzyme variants.

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Section: Cell-free Protein Synthesismentioning

confidence: 99%

Application of Cell-Free Protein Synthesis for Faster Biocatalyst Development

2019

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“…Cell-free systems are typically composed from the minimally-prepared extracts of lysed cells or reconstituted from purified biochemical components, supplemented with the desired DNA template and a chemical mixture to supply energy, amino acids, and small molecules to the system. A recent review of current organisms, methods, and applications is offered by [3].…”

Section: List Of Figuresmentioning

confidence: 99%

“…Anecdotally, plasmid DNA extracted from cells using various commercial kits give different and inconsistent results when used in cell-free systems. Workshop participants report that midi and maxi prep kits [36][37][38] 3 , although more time consuming than mini prep kits [38,39] 3 , generally yield DNA templates that perform better for protein production using cell-free systems than mini prep kits, which require additional purification steps for DNA templates of suitable quality. The Murray laboratory [4] improves the quality of DNA templates after extraction through three consecutive ethanol washes, while the Noireaux laboratory [40] purifies DNA templates after extraction with a cleanup kit developed for use with polymerase chain reaction (PCR) products to obtain a higher protein yield relative to that same DNA template prepared without the additional purification step [41].…”

Section: Dna Template Preparation Working Group Led By Vincent Noirementioning

confidence: 99%

CELL-FREE (comparable engineered living lysates for research education and entrepreneurship) workshop report

Romantseva¹,

Strychalski²

2020

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Publications in the SP1500 subseries are intended to capture external perspectives related to NIST standards, measurement, and testing-related efforts. These external perspectives can come from industry, academia, government, and others. These reports are intended to document external perspectives and do not represent official NIST positions. The opinions, recommendations, findings, and conclusions in this publication do not necessarily reflect the views or policies of NIST or the United States Government.

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“…Cell-free protein synthesis (CFPS) systems are emerging as effective platforms for in vitro synthetic biology and biotechnology applications from fundamental research to biomanufacturing (Carlson et al, 2012;Bundy et al, 2018;Li et al, 2018b;Swartz, 2018;Khambhati et al, 2019;Liu et al, 2019;Silverman et al, 2020). Such platforms separate the cell growth and the protein synthesis into two stages, which can alleviate the cell's metabolic burden and enhance the productivity.…”

Section: Introductionmentioning

confidence: 99%

Establishing a Eukaryotic Pichia pastoris Cell-Free Protein Synthesis System

Zhang

Liu

2020

Front. Bioeng. Biotechnol.

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In recent years, cell-free protein synthesis (CFPS) systems have been used to synthesize proteins, prototype genetic elements, manufacture chemicals, and diagnose diseases. These exciting, novel applications lead to a new wave of interest in the development of new CFPS systems that are derived from prokaryotic and eukaryotic organisms. The eukaryotic Pichia pastoris is emerging as a robust chassis host for recombinant protein production. To expand the current CFPS repertoire, we report here the development and optimization of a eukaryotic CFPS system, which is derived from a protease-deficient strain P. pastoris SMD1163. By developing a simple crude extract preparation protocol and optimizing CFPS reaction conditions, we were able to achieve superfolder green fluorescent protein (sfGFP) yields of 50.16 ± 7.49 µg/ml in 5 h batch reactions. Our newly developed P. pastoris CFPS system fits to the range of the productivity achieved by other eukaryotic CFPS platforms, normally ranging from several to tens of micrograms protein per milliliter in batch mode reactions. Looking forward, we believe that our P. pastoris CFPS system will not only expand the CFPS toolbox for synthetic biology applications, but also provide a novel platform for cost-effective, high-yielding production of complex proteins that need post-translational modification and functionalization.

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Cell-free protein synthesis: Recent advances in bacterial extract sources and expanded applications

Cited by 57 publications

References 103 publications

Application of Cell-Free Protein Synthesis for Faster Biocatalyst Development

Application of Cell-Free Protein Synthesis for Faster Biocatalyst Development

CELL-FREE (comparable engineered living lysates for research education and entrepreneurship) workshop report

Establishing a Eukaryotic Pichia pastoris Cell-Free Protein Synthesis System

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