Cell-Free Protein Synthesis

TierraBlog-003-Cell-FreeSynthesis.pdf

Khambhati K, Bhattacharjee G, Gohil N, Braddick D, Kulkarni V, Singh V. Exploring the Potential of Cell-Free Protein Synthesis for Extending the Abilities of Biological Systems. Front Bioeng Biotechnol. 2019;7. doi:10.3389/fbioe.2019.00248

Irastortza-Olaziregi M, Amster-Choder O. Coupled Transcription-Translation in Prokaryotes: An Old Couple With New Surprises.Front Microbiol. 2020;11:624830.doi:10.3389/fmicb.2020.624830

McGary K, Nudler E. RNA polymerase and the ribosome: the close relationship. Curr Opin Microbiol. 2013;16(2):112-117.doi:10.1016/j.mib.2013.01.010

Nirenberg MW, Matthaei JH. The dependence of cell-free protein synthesis in E. coli upon naturally occurring or synthetic polyribonucleotides. Proc Natl Acad Sci U S A. 1961;47(10):1588-1602. doi:10.1073/pnas.47.10.1588

Hunt AC, Vögeli B, Hassan AO, et al. A rapid cell-free expression and screening platform for antibody discovery. Nat Commun. 2023;14(1):3897. doi:10.1038/s41467-023-38965-w

Ramm F, Kaser D, König I, et al. Synthesis of biologically active Shiga toxins in cell-free systems. Sci Rep. 2024;14(1):6043.doi:10.1038/s41598-024-56190-3

 Failmezger J, Scholz S, Blombach B, Siemann-Herzberg M. Cell-Free Protein Synthesis From Fast-Growing Vibrio natriegens. Front Microbiol. 2018;9. doi:10.3389/fmicb.2018.01146

Wiegand DJ, Lee HH, Ostrov N, Church GM. Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens. JoVE (Journal of Visualized Experiments). 2019;(145):e59495. doi:10.3791/59495

Ji X, Liu WQ, Li J. Recent advances in applying cell-free systems for high-value and complex natural product biosynthesis. Current Opinion in Microbiology. 2022;67:102142. doi:10.1016/j.mib.2022.102142

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Li J, Zhang L, Liu W. Cell-free synthetic biology for in vitro biosynthesis of pharmaceutical natural products. Synth Syst Biotechnol. 2018;3(2):83-89. doi:10.1016/j.synbio.2018.02.002

SUN ZZ, Robertson DE, TREGO KS, CHIAO AC, IV LEM. Anaerobic cell-free systems and environments and methods for making and using same. Published online August 26, 2020. Accessed May 24, 2024. https://patents.google.com/patent/EP3697803A2/en

Cole SD, Miklos AE, Chiao AC, Sun ZZ, Lux MW. Methodologies for preparation of prokaryotic extracts for cell-free expression systems. Synthetic and Systems Biotechnology. 2020;5(4):252-267. doi:10.1016/j.synbio.2020.07.006

Macek B, Forchhammer K, Hardouin J, Weber-Ban E, Grangeasse C, Mijakovic I. Protein post-translational modifications in bacteria.Nat Rev Microbiol. 2019;17(11):651-664. doi:10.1038/s41579-019-0243-0

Ezure T, Nanatani K, Sato Y, et al. A Cell-Free Translocation System Using Extracts of Cultured Insect Cells to Yield Functional Membrane Proteins. PLOS ONE. 2014;9(12):e112874. doi:10.1371/journal.pone.0112874

Quast RB, Kortt O, Henkel J, et al. Automated production of functional membrane proteins using eukaryotic cell-free translation systems. Journal of Biotechnology. 2015;203:45-53. doi:10.1016/j.jbiotec.2015.03.015

Harbers M. Wheat germ systems for cell-free protein expression. FEBS Letters. 2014;588(17):2762-2773. doi:10.1016/j.febslet.2014.05.061

Scheele G, Blackburn P. Role of mammalian RNase inhibitor in cell-free protein synthesis. Proc Natl Acad Sci U S A.1979;76(10):4898-4902. doi:10.1073/pnas.76.10.4898

Marcu K, Dudock B. Characterization of a highly efficient protein synthesizing system derived from commercial wheat germ. Nucleic Acids Res. 1974;1(11):1385-1397. doi:10.1093/nar/1.11.1385

Goshima N, Kawamura Y, Fukumoto A, et al. Human protein factory for converting the transcriptome into an in vitro-expressed proteome,. Nat Methods. 2008;5(12):1011-1017. doi:10.1038/nmeth.1273

Arumugam TU, Ito D, Takashima E, et al. Application of wheat germ cell-free protein expression system for novel malaria vaccine candidate discovery. Expert Rev Vaccines. 2014;13(1):75-85. doi:10.1586/14760584.2014.861747

Buntru M, Vogel S, Spiegel H, Schillberg S. Tobacco BY-2 cell-free lysate: an alternative and highly-productive plant-based in vitro translation system. BMC Biotechnology. 2014;14(1):37. doi:10.1186/1472-6750-14-37

Mikami S, Kobayashi T, Masutani M, Yokoyama S, Imataka H. A human cell-derived in vitro coupled transcription/translation system optimized for production of recombinant proteins. Protein Expr Purif. 2008;62(2):190-198. doi:10.1016/j.pep.2008.09.002

Rosenblum G, Cooperman BS. Engine out of the Chassis: Cell-Free Protein Synthesis and its Uses. FEBS Lett. 2014;588(2):261-268. doi:10.1016/j.febslet.2013.10.016

Mureev S, Kovtun O, Nguyen UTT, Alexandrov K. Species-independent translational leaders facilitate cell-free expression. Nat Biotechnol. 2009;27(8):747-752. doi:10.1038/nbt.1556

Shinoda T, Shinya N, Ito K, et al. Cell-free methods to produce structurally intact mammalian membrane proteins. Sci Rep. 2016;6(1):30442. doi:10.1038/srep30442

Salehi ASM, Smith MT, Bennett AM, Williams JB, Pitt WG, Bundy BC. Cell-free protein synthesis of a cytotoxic cancer therapeutic: Onconase production and a just-add-water cell-free system. Biotechnol J. 2016;11(2):274-281. doi:10.1002/biot.201500237

Porche KB, Lanclos CE, Kwon YC. Challenging Post-translational Modifications in the Cell-free Protein Synthesis System. Synthetic Biology and Engineering. 2023;1(2):10011. doi:10.35534/sbe.2023.10011

Martin RW, Des Soye BJ, Kwon YC, et al. Cell-free protein synthesis from genomically recoded bacteria enables multisite incorporation of noncanonical amino acids. Nat Commun. 2018;9(1):1203. doi:10.1038/s41467-018-03469-5

Gupta MD, Flaskamp Y, Roentgen R, et al. Scaling eukaryotic cell-free protein synthesis achieved with the versatile and highyielding tobacco BY-2 cell lysate. Biotechnology and Bioengineering. 2023;120(10):2890-2906. doi:10.1002/bit.28461

Silverman AD, Karim AS, Jewett MC. Cell-free gene expression: an expanded repertoire of applications. Nat Rev Genet. 2020;21(3):151-170. doi:10.1038/s41576-019-0186-3

Prokaryotic and eukaryotic systems have unique advantages and disadvantages for CFPS protocols. Prokaryotes are cost-effective with high growth rates, but eukaryotic systems can perform post-translational modifications despite their higher cost.

Successful CFPS requires understanding protein types and factors affecting yields. Different proteins require specific reagents and conditions to optimize transcription efficiency and maximize protein yields.

Advances in strain engineering, such as using engineered strains of E. coli, enhance the production of biologics through CFPS.

Selecting a Cell-Free Protein Synthesis System

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Gupta V, Sengupta M, Prakash J, Tripathy BC. Production of Recombinant Pharmaceutical Proteins. Basic and Applied Aspects of Biotechnology. Published online October 23, 2016:77-101. doi:10.1007/978-981-10-0875-7_4

Gregorio NE, Levine MZ, Oza JP. A User’s Guide to Cell-Free Protein Synthesis. Methods Protoc. 2019;2(1):24. doi:10.3390/mps2010024

Zhang L, Lin X, Wang T, Guo W, Lu Y. Development and comparison of cell-free protein synthesis systems derived from typical bacterial chassis. Bioresources and Bioprocessing. 2021;8(1):58. doi:10.1186/s40643-021-00413-2

Schoborg JA, Hodgman CE, Anderson MJ, Jewett MC. Substrate replenishment and byproduct removal improve yeast cell-free protein synthesis. Biotechnol J. 2014;9(5):630-640. doi:10.1002/biot.201300383

Garamella J, Marshall R, Rustad M, Noireaux V. The All E. coli TX-TL Toolbox 2.0: A Platform for Cell-Free Synthetic Biology. ACS Synth Biol. 2016;5(4):344-355. doi:10.1021/acssynbio.5b00296

Yim SS, Johns NI, Noireaux V, Wang HH. Protecting Linear DNA Templates in Cell-Free Expression Systems from Diverse Bacteria. ACS Synth Biol. 2020;9(10):2851-2855. doi:10.1021/acssynbio.0c00277

Carlson ED, Gan R, Hodgman CE, Jewett MC. Cell-Free Protein Synthesis: Applications Come of Age. Biotechnol Adv. 2012;30(5):1185-1194. doi:10.1016/j.biotechadv.2011.09.016

Groff D, Carlos NA, Chen R, et al. Development of an E. coli strain for cell‐free ADC manufacturing. Biotechnol Bioeng. 2022;119(1):162-175. doi:10.1002/bit.27961

Zawada JF, Yin G, Steiner AR, et al. Microscale to manufacturing scale-up of cell-free cytokine production--a new approach for shortening protein production development timelines. Biotechnol Bioeng. 2011;108(7):1570-1578. doi:10.1002/bit.23103

He W, Felderman M, Evans AC, et al. Cell-free production of a functional oligomeric form of a Chlamydia major outer-membrane protein (MOMP) for vaccine development. J Biol Chem. 2017;292(36):15121-15132. doi:10.1074/jbc.M117.784561

Jin X, Kightlinger W, Kwon YC, Hong SH. Rapid production and characterization of antimicrobial colicins using Escherichia coli-based cell-free protein synthesis. Synthetic Biology. 2018;3(1):ysy004. doi:10.1093/synbio/ysy004

Makurvet FD. Biologics vs. small molecules: Drug costs and patient access. Medicine in Drug Discovery. 2021;9:100075. doi:10.1016/j.medidd.2020.100075

Dondapati, S.K., Stech, M., Zemella, A. et al. Cell-Free Protein Synthesis: A Promising Option for Future Drug Development. BioDrugs 34, 327–348 (2020). https://doi.org/10.1007/s40259-020-00417-y

Proteins are vital for advancing an array of biotechnological applications, including drug development.

Traditional cell-based synthesis methods face challenges with scalability, high costs, and restrictions on producing complex proteins.

Cell-free protein synthesis (CFPS) overcomes these limitations by enabling efficient protein production without cellular constraints.

The Tierra Protein Platform utilizes AI and automation to rapidly synthesize a diverse range of proteins, enhancing research and development efforts.

Breaking the bounds of biology: innovations in cell-free protein synthesis

Therapeutics

TierraBlog-001_FromTargetstoTreatment.pdf

Evolving strategies and application of proteins and peptide therapeutics in cancer treatment. Biomed. Pharmacother. 163, 114832 (2023).

Seebacher, N. A., Stacy, A. E., Porter, G. M. & Merlot, A. M. Clinical development of targeted and immune based anti-cancer therapies. J. Exp. Clin. Cancer Res. 38, 1–39 (2019).

Rosano, G. L., Morales, E. S. & Ceccarelli, E. A. New tools for recombinant protein production in Escherichia coli: A 5-year update. Protein Sci. 28, 1412–1422 (2019).

Lu, R.-M. et al. Development of therapeutic antibodies for the treatment of diseases. J. Biomed. Sci. 27, 1–30 (2020).

Lv, D. et al. CancerProteome: a resource to functionally decipher the proteome landscape in cancer. Nucleic Acids Res. 52, D1155–D1162 (2024).

Hamerlik, P. et al. Autocrine VEGF-VEGFR2-Neuropilin-1 signaling promotes glioma stem-like cell viability and tumor growth. J. Exp. Med. 209, 507–520 (2012).

Cieslewicz, M. et al. Targeted delivery of proapoptotic peptides to tumor-associated macrophages improves survival. Proc. Natl. Acad. Sci. U. S. A. 110, 15919–15924 (2013).

Granier, C. et al. Mechanisms of action and rationale for the use of checkpoint inhibitors in cancer. ESMO Open 2, e000213 (2017).

Mikalsen, T., Gerits, N. & Moens, U. Inhibitors of signal transduction protein kinases as targets for cancer therapy. Biotechnol. Annu. Rev. 12, 153–223 (2006).

Dondapati, S. K., Stech, M., Zemella, A. & Kubick, S. Cell-Free Protein Synthesis: A Promising Option for Future Drug Development. BioDrugs 34, 327–348 (2020).

Kintzing, J. R., Filsinger Interrante, M. V. & Cochran, J. R. Emerging Strategies for Developing Next-Generation Protein Therapeutics for Cancer Treatment. Trends Pharmacol. Sci. 37, 993–1008 (2016).

Soon, T. N., Chia, A. Y. Y., Yap, W. H. & Tang, Y.-Q. Anticancer Mechanisms of Bioactive Peptides. Protein Pept. Lett. 27, 823–830 (2020).

Xie, M., Liu, D. & Yang, Y. Anti-cancer peptides: classification, mechanism of action, reconstruction and modification. Open Biol. 10, 200004 (2020).

Wang, L. et al. Therapeutic peptides: current applications and future directions. Signal Transduct. Target. Ther. 7, (2022).

Antibody therapeutics product data. The Antibody Society https://www.antibodysociety.org/antibody-therapeutics-product-data/ (2021).

Sifniotis, V., Cruz, E., Eroglu, B. & Kayser, V. Current Advancements in Addressing Key Challenges of Therapeutic Antibody Design, Manufacture, and Formulation. Antibodies 8, 36 (2019).

Madani, A. et al. Large language models generate functional protein sequences across diverse families. Nat. Biotechnol. 41, 1099–1106 (2023).

The cancer proteome, with its remarkable diversity, encompasses a vast array of proteins distinguished by their functions and cellular locations, underscoring the multifaceted nature of cancer biology.

Targeted protein-based therapies, particularly those centered around antibodies, are on the rise, yet they encounter significant hurdles in terms of developability and manufacturability.

Pioneering advancements in gene expression and protein engineering are catalyzing a revolution in the generation of personalized and more efficacious protein-based cancer treatments.

From targets to treatments: insights from the cancer proteome

Experience AI-driven custom protein synthesis at scale, only from Tierra.

Discover the world of protein-powered cell-free research.