ArticleJournal of biochemistry2017
A reproducible and scalable procedure for preparing bacterial extracts for cell-free protein synthesis.
Article in Journal of biochemistry, 2017. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 28 papers.
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Who cites it
28 citing papers in PubMed.
- Structural basis for auto-inhibition of the Rac1/Cdc42 guanine nucleotide exchange factor DOCK6 by oligomer formation.Communications biology · 2026Article
- Conformational alteration of DOCK5•ELMO1 signalosome on lipid membrane.Communications biology · 2025Article
- Standardizing Bacterial Extracellular Vesicle Purification: A Call for Consensus.Journal of microbiology and biotechnology · 2025Review
- Cell-free protein synthesis and vesicle systems for programmable therapeutic manufacturing and delivery.Journal of biological engineering · 2025Review
- RhoG facilitates a conformational transition in the guanine nucleotide exchange factor complex DOCK5/ELMO1 to an open state.The Journal of biological chemistry · 2024Article
- Structural analyses of the GI.4 norovirus by cryo-electron microscopy and X-ray crystallography revealing binding sites for human monoclonal antibodies.Journal of virology · 2024Article
- Efficiency of transcription and translation of cell-free protein synthesis systems in cell-sized lipid vesicles with changing lipid composition determined by fluorescence measurements.Scientific reports · 2024Article
- Cyanorhodopsin-II represents a yellow-absorbing proton-pumping rhodopsin clade within cyanobacteria.The ISME journal · 2024Article
- Preparing for the future of precision medicine: synthetic cell drug regulation.Synthetic biology (Oxford, England) · 2024Review
- Designing of an extract production protocol for industrial application of cell-free protein synthesis technology: Building from a current best practice to a quality by design approach.Engineering biology · 2023Review
- Opportunities to accelerate extracellular vesicle research with cell-free synthetic biology.Journal of extracellular biology · 2023Article
- Targeting Ras-binding domain of ELMO1 by computational nanobody design.Communications biology · 2023Article
- A specific G9a inhibitor unveils BGLT3 lncRNA as a universal mediator of chemically induced fetal globin gene expression.Nature communications · 2023Article
- Potent SARS-CoV-2 neutralizing antibodies with therapeutic effects in two animal models.iScience · 2022Article
- Crystal Structure of an Archaeal Tyrosyl-tRNA Synthetase Bound to Photocaged L-Tyrosine and Its Potential Application to Time-Resolved X-ray Crystallography.International journal of molecular sciences · 2022Article
- Lewis fucose is a key moiety for the recognition of histo-blood group antigens by GI.9 norovirus, as revealed by structural analysis.FEBS open bio · 2022Article
- Key reaction components affect the kinetics and performance robustness of cell-free protein synthesis reactions.Computational and structural biotechnology journal · 2022Article
- Identification of TCR repertoires in functionally competent cytotoxic T cells cross-reactive to SARS-CoV-2.Communications biology · 2021Article
- Reduced efficacy of a Src kinase inhibitor in crowded protein solution.Nature communications · 2021Article
- Cryo-EM structure of the human ELMO1-DOCK5-Rac1 complex.Science advances · 2021Article
Corrections and comments
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Authors and funding
9 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Cell-free protein synthesis is a useful method for preparing proteins for functional or structural analyses. However, batch-to-batch variability with regard to protein synthesis activity remains a problem for large-scale production of cell extract in the laboratory. To address this issue, we have developed a novel procedure for large-scale preparation of bacterial cell extract with high protein synthesis activity. The developed procedure comprises cell cultivation using a fermentor, harvesting and washing of cells by tangential flow filtration, cell disruption with high-pressure homogenizer and continuous diafiltration. By optimizing and combining these methods, ∼100 ml of the cell extract was prepared from 150 g of Escherichia coli cells. The protein synthesis activities, defined as the yield of protein per unit of absorbance at 260 nm of the cell extract, were shown to be reproducible, and the average activity of several batches was twice that obtained using a previously reported method. In addition, combinatorial use of the high-pressure homogenizer and diafiltration increased the scalability, indicating that the cell concentration at disruption varies from 0.04 to 1 g/ml. Furthermore, addition of Gam protein and examinations of the N-terminal sequence rendered the extract prepared here useful for rapid screening with linear DNA templates.
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