High-throughput preparation methods of crude extract for robust cell-free protein synthesis

Kwon, Yong Chan; Jewett, Michael C.

doi:10.1038/srep08663

Cited by 329 publications

(516 citation statements)

References 57 publications

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“…We tested protein synthesis yields in 20 h batch reactions across three different E. coli crude extracts (S30 crude extracts). 33 On the basis of the expression yields of GrsA and GrsB1, we found that E. coli BL21 Star (DE3) extract synthesized the highest amount of NRPS, yielding full-length, soluble GrsA at ~106 µg/mL and GrsB1 at ~77 µg/mL (Figure S1). Fully assembled GrsA (126 kDa) and GrsB1 (121 kDa) with correct molecular weight bands were observed by SDS-PAGE and further confirmed by autoradiogram analysis (Figure S2).…”

Section: Resultsmentioning

confidence: 97%

In Vitro Reconstruction of Nonribosomal Peptide Biosynthesis Directly from DNA Using Cell-Free Protein Synthesis

Goering

McClure

et al. 2016

ACS Synth. Biol.

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Genome sequencing has revealed that a far greater number of natural product biosynthetic pathways exist than there are known natural products. To access these molecules directly and deterministically, a new generation of heterologous expression methods is needed. Cell-free protein synthesis has not previously been used to study nonribosomal peptide biosynthesis, and provides a tunable platform with advantages over conventional methods for protein expression. Here, we demonstrate the use of cell-free protein synthesis to biosynthesize a cyclic dipeptide with correct absolute stereochemistry. From a single-pot reaction, we measured the expression of two nonribosomal peptide synthetases larger than 100 kDa, and detected high-level production of a diketopiperazine. Using quantitative LC–MS and synthetically prepared standard, we observed production of this metabolite at levels higher than previously reported from cell-based recombinant expression, approximately 12 mg/L. Overall, this work represents a first step to apply cell-free protein synthesis to discover and characterize new natural products.

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Section: Resultsmentioning

confidence: 97%

In Vitro Reconstruction of Nonribosomal Peptide Biosynthesis Directly from DNA Using Cell-Free Protein Synthesis

Goering

McClure

et al. 2016

ACS Synth. Biol.

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“…In recent years, systematic optimization of each step in extract preparation for E. coli CFPS has led to more robust and productive extracts (Carlson et al, 2012;Kwon and Jewett, 2015). In recent years, systematic optimization of each step in extract preparation for E. coli CFPS has led to more robust and productive extracts (Carlson et al, 2012;Kwon and Jewett, 2015).…”

Section: Optimization Of Cell Extract Preparationmentioning

confidence: 99%

Establishing a high yielding streptomyces‐based cell‐free protein synthesis system

Wang

Kwon

et al. 2017

Biotech & Bioengineering

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Cell-free protein synthesis (CFPS) has emerged as a powerful platform for applied biotechnology and synthetic biology, with a range of applications in synthesizing proteins, evolving proteins, and prototyping genetic circuits. To expand the current CFPS repertoire, we report here the development and optimization of a Streptomyces-based CFPS system for the expression of GC-rich genes. By developing a streamlined crude extract preparation protocol and optimizing reaction conditions, we were able to achieve active enhanced green fluorescent protein (EGFP) yields of greater than 50 μg/mL with batch reactions lasting up to 3 h. By adopting a semi-continuous reaction format, the EGFP yield could be increased to 282 ± 8 μg/mL and the reaction time was extended to 48 h. Notably, our extract preparation procedures were robust to multiple Streptomyces lividans and Streptomyces coelicolor strains, although expression yields varied. We show that our optimized Streptomyces lividans system provides benefits when compared to an Escherichia coli-based CFPS system for increasing percent soluble protein expression for four Streptomyces-originated high GC-content genes that are involved in biosynthesis of the nonribosomal peptides tambromycin and valinomycin. Looking forward, we believe that our Streptomyces-based CFPS system will contribute significantly towards efforts to express complex natural product gene clusters (e.g., nonribosomal peptides and polyketides), providing a new avenue for obtaining and studying natural product biosynthesis pathways. Biotechnol. Bioeng. 2017;114: 1343-1353. © 2017 Wiley Periodicals, Inc.

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“…Traditionally, the established preparation procedures require complex protocols, special experience, and expensive equipment for mechanical cell disruption (French press or bead beater) that is not readily available in most laboratories and that limits the throughput in terms of sample size and numbers (reviewed in ref. (14 – 16)). Achieving reproducible results using these methods also requires considerable experience, limiting access to the technology.…”

Section: Introductionmentioning

confidence: 99%

Rapid and Scalable Preparation of Bacterial Lysates for Cell-Free Gene Expression

et al. 2017

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Cell-free gene expression systems are emerging as an important platform for a diverse range of synthetic biology and biotechnology applications, including production of robust field-ready biosensors. Here, we combine programmed cellular autolysis with a freeze-thaw or freeze-dry cycle to create a practical, reproducible, and a labor- and cost-effective approach for rapid production of bacterial lysates for cell-free gene expression. Using this method, robust and highly active bacterial cell lysates can be produced without specialized equipment at a wide range of scales, making cell-free gene expression easily and broadly accessible. Moreover, live autolysis strain can be freeze-dried directly and subsequently lysed upon rehydration to produce active lysate. We demonstrate the utility of autolysates for synthetic biology by regulating protein production and degradation, implementing quorum sensing, and showing quantitative protection of linear DNA templates by GamS protein. To allow versatile and sensitive β-galactosidase (LacZ) based readout we produce autolysates with no detectable background LacZ activity and use them to produce sensitive mercury(II) biosensors with LacZ-mediated colorimetric and fluorescent outputs. The autolysis approach can facilitate wider adoption of cell-free technology for cell-free gene expression as well as other synthetic biology and biotechnology applications, such as metabolic engineering, natural product biosynthesis, or proteomics.

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High-throughput preparation methods of crude extract for robust cell-free protein synthesis

Cited by 329 publications

References 57 publications

In Vitro Reconstruction of Nonribosomal Peptide Biosynthesis Directly from DNA Using Cell-Free Protein Synthesis

In Vitro Reconstruction of Nonribosomal Peptide Biosynthesis Directly from DNA Using Cell-Free Protein Synthesis

Establishing a high yielding streptomyces‐based cell‐free protein synthesis system

Rapid and Scalable Preparation of Bacterial Lysates for Cell-Free Gene Expression

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