Abstract
Cell-free extracts have been researched and continuously streamlined for around 50 years. It is believed that these extracts work best when routinely obtained from exponentially growing cells to capture the most active translation system. Here we report on an active cell-free extract derived from E. coli A19 that was harvested under nongrowing, stressed conditions. Although this process is based on the conventional routine process for the production of S30-extracts, our process is less labor intensive and reduces variability between extracts.
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References
Nirenberg MW, Matthaei JH (1961) The dependence of cell-free protein synthesis in E. coli upon naturally occurring or synthetic polyribonucleotides. Proc Natl Acad Sci U S A 47(10):1588–1602. https://doi.org/10.1073/pnas.47.10.1588
Zubay G (1973) In vitro synthesis of protein in microbial systems. Annu Rev Genet 7(1):267–287. https://doi.org/10.1146/annurev.ge.07.120173.001411
Cole SD, Miklos AE, Chiao AC, Sun ZZ, Lux MW (2020) Methodologies for preparation of prokaryotic extracts for cell-free expression systems. Synth Syst Biotechnol 5(4):252–267. https://doi.org/10.1016/j.synbio.2020.07.006
Silverman AD, Karim AS, Jewett MC (2020) Cell-free gene expression: an expanded repertoire of applications. Nat Rev Genet 21(3):151–170. https://doi.org/10.1038/s41576-019-0186-3
Dennis PP, Bremer H (2008) Modulation of chemical composition and other parameters of the cell at different exponential growth rates. EcoSal Plus 3(1). https://doi.org/10.1128/ecosal.5.2.3
Wiegand DJ, Lee HH, Ostrov N, Church GM (2018) Establishing a cell-free vibrio natriegens expression system. ACS Synth Biol 7(10):2475–2479. https://doi.org/10.1021/acssynbio.8b00222
Des Soye BJ, Davidson SR, Weinstock MT, Gibson DG, Jewett MC (2018) Establishing a high-yielding cell-free protein synthesis platform derived from vibrio natriegens. ACS Synth Biol 7(9):2245–2255. https://doi.org/10.1021/acssynbio.8b00252
Failmezger J, Scholz S, Blombach B, Siemann-Herzberg M (2018) Cell-free protein synthesis from fast-growing Vibrio natriegens. Front Microbiol 9:1–10. https://doi.org/10.3389/fmicb.2018.01146
Failmezger J, Rauter M, Nitschel R, Kraml M, Siemann-Herzberg M (2017) Cell-free protein synthesis from non-growing, stressed Escherichia coli. Sci Rep 7(1):16524. https://doi.org/10.1038/s41598-017-16767-7
Dopp BJL, Tamiev DD, Reuel NF (2019) Cell-free supplement mixtures: elucidating the history and biochemical utility of additives used to support in vitro protein synthesis in E. coli extract. Biotechnol Adv 37(1):246–258. https://doi.org/10.1016/j.biotechadv.2018.12.006
Wegerer A, Sun T, Altenbuchner J (2008) Optimization of an E. coli L-rhamnose-inducible expression vector: test of various genetic module combinations. BMC Biotechnol 8:1–12. https://doi.org/10.1186/1472-6750-8-2
Kim J, Copeland CE, Padumane SR, Kwon YC (2019) A crude extract preparation and optimization from a genomically engineered Escherichia coli for the cell-free protein synthesis system: practical laboratory guideline. Methods Protoc 2(3):1–15. https://doi.org/10.3390/mps2030068
Levine MZ et al (2020) Activation of energy metabolism through growth media reformulation enables a 24-h workflow for cell-free expression. ACS Synth Biol 9(10):2765–2774. https://doi.org/10.1021/acssynbio.0c00283
Liu DV, Zawada JF, Swartz JR (2005) Streamlining Escherichia Coli S30 extract preparation for economical cell-free protein synthesis. Biotechnol Prog 21(2):460–465. https://doi.org/10.1021/bp049789y
Acknowledgments
This work was supported by the Bundesministerium für Bildung und Forschung, BMBF, Berlin, Germany, BMBF grant number 031A157D. We thank Dinesh Pinto for helpful advice on the manuscript.
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Hiering, F., Failmezger, J., Siemann-Herzberg, M. (2022). Preparation and Screening of Cell-Free Extract from Nongrowing Escherichia coli A19 Cells. In: Karim, A.S., Jewett, M.C. (eds) Cell-Free Gene Expression. Methods in Molecular Biology, vol 2433. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1998-8_3
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DOI: https://doi.org/10.1007/978-1-0716-1998-8_3
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Online ISBN: 978-1-0716-1998-8
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