Preparation and Screening of Cell-Free Extract from Nongrowing Escherichia coli A19 Cells

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.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Subscribe and save

• Get 10 units per month
• Download Article/Chapter or eBook
• 1 Unit = 1 Article or 1 Chapter
• Cancel anytime

Buy Now

eBook EUR 128.39 Price includes VAT (France)

Softcover Book EUR 163.51 Price includes VAT (France)

Hardcover Book EUR 232.09 Price includes VAT (France)

Tax calculation will be finalised at checkout

Purchases are for personal use only

Similar content being viewed by others

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

Cell-free protein synthesis from non-growing, stressed Escherichia coli

Detergent-assisted Enhancement of the Translation Rate during Cell-free Synthesis of Peptides in an Escherichia coli Extract

References

1. 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.47.10.1588ArticleCASPubMedPubMed CentralGoogle Scholar
2. 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.07.120173.001411ArticleCASPubMedGoogle Scholar
3. 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.2020.07.006ArticlePubMedPubMed CentralGoogle Scholar
4. 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/s41576-019-0186-3ArticleCASPubMedGoogle Scholar
5. 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
6. 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.8b00222ArticleCASPubMedGoogle Scholar
7. 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.8b00252ArticleCASPubMedGoogle Scholar
8. 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.2018.01146ArticleGoogle Scholar
9. 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/s41598-017-16767-7ArticleCASPubMedPubMed CentralGoogle Scholar
10. 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.2018.12.006ArticleCASPubMedGoogle Scholar
11. 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/10.1186/1472-6750-8-2ArticleCASGoogle Scholar
12. 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/mps2030068ArticleCASGoogle Scholar
13. 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.0c00283ArticleCASPubMedGoogle Scholar
14. 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/bp049789yArticleCASPubMedGoogle Scholar

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.

Author information

Authors and Affiliations

1. Institute of Biochemical Engineering, Universität Stuttgart, Stuttgart, Germany Florian Hiering, Jurek Failmezger & Martin Siemann-Herzberg

1. Florian Hiering