Cell-free biomanufacturing

Bundy, Bradley C.; Hunt, J. Porter; Jewett, Michael C.; Swartz, James R.; Wood, David; Frey, Douglas D.; Rao, Govind

doi:10.1016/j.coche.2018.10.003

Cited by 66 publications

(49 citation statements)

References 66 publications

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“…For example, multiple labs have removed the native tRNAs and then added a minimal set of in vitro synthesized tRNAs, essentially emancipating most codons for competition-free ncAA incorporation [63, 132]. Additional advantages that in vitro or ‘cell-free’ protein synthesis provides over in vivo expression include direct access to the reaction environment, eliminating transport limitations of ncAAs across cell membranes and walls and allowing facile supplementation with exogenous components to improve incorporation efficiency [69, 133]. The flexibility of this system allows the incorporation of less-soluble ncAAs with click-compatible side chains, expanding the repertoire for protein labeling [133].…”

Section: Future Directionsmentioning

confidence: 99%

“…Additional advantages that in vitro or ‘cell-free’ protein synthesis provides over in vivo expression include direct access to the reaction environment, eliminating transport limitations of ncAAs across cell membranes and walls and allowing facile supplementation with exogenous components to improve incorporation efficiency [69, 133]. The flexibility of this system allows the incorporation of less-soluble ncAAs with click-compatible side chains, expanding the repertoire for protein labeling [133]. Importantly, cell-free systems can also be lyophilized for on-demand distributed use in an endotoxin-free format for point-of-care medicinal applications or for rapid response to market demands for biochemical products [134, 135].…”

Section: Future Directionsmentioning

confidence: 99%

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Non-canonical amino acid labeling in proteomics and biotechnology

et al. 2019

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Metabolic labeling of proteins with non-canonical amino acids (ncAAs) provides unique bioorthogonal chemical groups during de novo synthesis by taking advantage of both endogenous and heterologous protein synthesis machineries. Labeled proteins can then be selectively conjugated to fluorophores, affinity reagents, peptides, polymers, nanoparticles or surfaces for a wide variety of downstream applications in proteomics and biotechnology. In this review, we focus on techniques in which proteins are residue- and site-specifically labeled with ncAAs containing bioorthogonal handles. These ncAA-labeled proteins are: readily enriched from cells and tissues for identification via mass spectrometry-based proteomic analysis; selectively purified for downstream biotechnology applications; or labeled with fluorophores for in situ analysis. To facilitate the wider use of these techniques, we provide decision trees to help guide the design of future experiments. It is expected that the use of ncAA labeling will continue to expand into new application areas where spatial and temporal analysis of proteome dynamics and engineering new chemistries and new function into proteins are desired.

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Section: Future Directionsmentioning

confidence: 99%

Section: Future Directionsmentioning

confidence: 99%

Non-canonical amino acid labeling in proteomics and biotechnology

et al. 2019

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“…CFE has even been put forth as a powerful educational tool to teach the fundamentals of biology [ [26] , [27] , [28] ]. In the past two years, several reviews have summarized various aspects of CFE research [ [2] , [3] , [4] , 16 , [29] , [30] , [31] , [32] ]; here we examine the methods used for the production of prokaryotic extracts for CFE, which to date does not exist in the literature. We anticipate that this work will be of utility to new entrants to the field seeking to understand the differences between the myriad protocols, the relative advantages and disadvantages of each step, and the rationale behind methodological choices.…”

Section: Introductionmentioning

confidence: 99%

Methodologies for preparation of prokaryotic extracts for cell-free expression systems

Cole¹,

Miklos²,

Chiao³

et al. 2020

Synthetic and Systems Biotechnology

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Cell-free systems that mimic essential cell functions, such as gene expression, have dramatically expanded in recent years, both in terms of applications and widespread adoption. Here we provide a review of cell-extract methods, with a specific focus on prokaryotic systems. Firstly, we describe the diversity of Escherichia coli genetic strains available and their corresponding utility. We then trace the history of cell-extract methodology over the past 20 years, showing key improvements that lower the entry level for new researchers. Next, we survey the rise of new prokaryotic cell-free systems, with associated methods, and the opportunities provided. Finally, we use this historical perspective to comment on the role of methodology improvements and highlight where further improvements may be possible.

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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 biomanufacturing

Cited by 66 publications

References 66 publications

Non-canonical amino acid labeling in proteomics and biotechnology

Non-canonical amino acid labeling in proteomics and biotechnology

Methodologies for preparation of prokaryotic extracts for cell-free expression systems

Establishing a Eukaryotic Pichia pastoris Cell-Free Protein Synthesis System

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