cliA cell-free system is an in vitro tool widely used to study biological reactions that happen within cells apart from a full cell system, thus reducing the complex interactions typically found when working in a whole cell. Subcellular fractions can be isolated by ultracentrifugation to provide molecular machinery that can be used in reactions in the absence of many of the other cellular components. Eukaryotic and prokaryotic cell internals have been used for creation of these simplified environments. These systems have enabled cell-free synthetic biology to emerge, providing control over what reaction is being examined, as well as its yield, and lessening the considerations otherwise invoked when working with more sensitive live cells.
Cell-free systems may be divided into two primary classifications: cell extract-based, which remove components from within a whole cell for external use, and purified enzyme-based, which use purified components of the molecules known to be involved in a given process. The cell extract-based type are susceptible to problems like quick degradation of components outside their host, as shown in a study by Kitaoka et al. where a cell-free translation system based on Escherichia coli (E. coli), of the cell extract-based type, had the mRNA template degrade very quickly and led to the halt of protein synthesis.
The methods of preparation vary between situations of both types of cell-free systems.
Nobel prize winner Eduard Buchner was arguably the first to present a cell-free system using yeast extracts, but since then alternative sources have been found. E. coli, wheat germ, and rabbit reticulocytes have all proven useful to create cell-free systems by extraction of their interior components. E. coli 30S extracts have been acquired, for example, by grinding the bacteria with alumina, followed by further cleaning. Similarly, wheat germ has been ground with acid-washed sand or powdered glass to open the cell membranes up. Rabbit reticulocytes have been lysed in a solution of MgCl2 and had the extract filtered away from the membranes by centrifugation.
Cell-free synthetic pathway biotransformation biosystems can be prepared by mixing a number of purified enzymes and coenzymes. For example, tightly coupled ribosomes, which are compact and highly active, have been extracted and refined from E. coli through sucrose-density-gradient centrifugation.
Cell-free synthetic pathway biotransformation biosystems are proposed as a new low-cost biomanufacturing platform compared to microbial fermentation used for thousands of years. Cell-free biosystems have several advantages suitable in industrial applications:
- Very high product yields are usually accomplished without the formation of by-products or the synthesis of cell mass. For example, with an synthetic enzyme pathway, from the reaction with starch and water
- C6H10O5 (l) + 7 H2O (l) → 12 H2 (g) + 6 CO2 (g),
- nearly 12 H2 has been produced per glucose unit of polysaccharides and water, three times of the theoretical yield of the best anaerobic hydrogen-producing microorganisms.
- In vitro biosystems can implement some biological reactions that living microbes or chemical catalysts cannot implement before. For example, beta-1,4-glucosidic bond linked cellulose can be converted to alpha-1,4-glucosidic bond linked starch by a mixture of intracellular and extracellular enzymes in a single reaction container.
- Enzymatic systems, without the barrier of cellular membrane, usually have faster reaction rates than microbial systems. For instance, enzymatic fuel cells usually have much higher power outputs than microbial fuel cells.
- Enzyme cocktails can tolerate toxic compounds better than microorganisms.
- Enzyme mixtures usually work under broad reaction conditions, such as high temperature, low pH, the presence of organic solvents or ionic liquids.
In vitro biosystems can be easily controlled and accessed without membranes. Notably, in work leading to a Nobel prize the Nirenberg and Matthaei experiment used a cell-free system, of the cell extract-based type, to incorporate chosen amino acids tagged radioactively into synthesized proteins with 30S extracted from E. coli. More recent studies, such as the study done by Spirin et al. with prokaryotic and eukaryotic version of their cell-free translation system, have also synthesized proteins with increased production, incorporating techniques like continuous flow to add materials and remove products. With such advances in yield, productivity applications have been expanded, such as the synthesis of fusion proteins to potentially serve as vaccines for B-cell lymphomas. Additionally, cell-free protein synthesis is becoming a new alternative choice for fast protein synthesis.
Engineering of metabolic processes have been achieved through cell-free systems. Bujara et al., for example, were able to use glycolytic network extracts, consisting of enzymes from E. coli that produced dihydroxyacetone phosphate, to analyze in real-time the metabolite concentrations while altering enzyme levels, with the end result of optimal production of dihydroxyacetone phosphate. Further, Calhoun and Swartz were able to use a glycolytic intermediate to fuel a cell-free system, enabling relatively inexpensive ATP generation compared to reagent usage in phosphoenolpyruvate reactions.
Unnatural amino acid incorporation
Cell-free systems have also been used to incorporate unnatural amino acids. Shimizu et al. were able to change a stop codon to a sense codon by omitting the RF1 release factor, indicating ability to insert desired amino acids in unnatural situations. This is of use in systems where working inside a cell is problematic, such as the process of amino acid metabolism preventing specific labelling of amino acids that would be useful in multidimensional NMR spectroscopy. Kigawa et al.were able to successfully label amino acids in a cell-free system where amino acid metabolism was no longer present, thus making such systems useful to NMR studies.
- Swartz, Jim (2006-07-01). "Developing cell-free biology for industrial applications". Journal of Industrial Microbiology and Biotechnology. 33 (7): 476–485. doi:10.1007/s10295-006-0127-y. ISSN 1367-5435. PMID 16761165.
- "MeSH Browser". meshb.nlm.nih.gov. Retrieved 2017-10-18.
- Gregorio, Nicole E.; Levine, Max Z.; Oza, Javin P. (2019). "A User's Guide to Cell-Free Protein Synthesis". Methods and Protocols. 2 (1): 24. doi:10.3390/mps2010024. PMC 6481089. PMID 31164605.
- Zemella, Anne; Thoring, Lena; Hoffmeister, Christian; Kubick, Stefan (2015-11-01). "Cell-Free Protein Synthesis: Pros and Cons of Prokaryotic and Eukaryotic Systems". ChemBioChem. 16 (17): 2420–2431. doi:10.1002/cbic.201500340. ISSN 1439-7633. PMC 4676933. PMID 26478227.
- Lu, Yuan (2017). "Cell-free synthetic biology: Engineering in an open world". Synthetic and Systems Biotechnology. 2 (1): 23–27. doi:10.1016/j.synbio.2017.02.003. PMC 5625795. PMID 29062958.
- Rollin, Joseph A.; Tam, Tsz Kin; Zhang, Y.-H. Percival (2013-06-21). "New biotechnology paradigm: cell-free biosystems for biomanufacturing". Green Chemistry. 15 (7): 1708. doi:10.1039/c3gc40625c. ISSN 1463-9270.
- Shimizu, Yoshihiro; Inoue, Akio; Tomari, Yukihide; Suzuki, Tsutomu; Yokogawa, Takashi; Nishikawa, Kazuya; Ueda, Takuya (2001-05-23). "Cell-free translation reconstituted with purified components". Nature Biotechnology. 19 (8): 751–755. doi:10.1038/90802. PMID 11479568.
- Kitaoka, Yoshihisa; Nishimura, Norihiro; Niwano, Mitsuru (1996). "Cooperativity of stabilized mRNA and enhanced translation activity in the cell-free system". Journal of Biotechnology. 48 (1–2): 1–8. doi:10.1016/0168-1656(96)01389-2. PMID 8818268.
- Barnett, James A.; Lichtenthaler, Frieder W. (15 March 2001). "A history of research on yeasts 3: Emil Fischer, Eduard Buchner and their contemporaries, 1880-1900". Yeast. 18 (4): 363–388. doi:10.1002/1097-0061(20010315)18:4<363::AID-YEA677>3.0.CO;2-R. ISSN 1097-0061.
- Swartz, James R. (2012-01-01). "Transforming biochemical engineering with cell-free biology". AIChE Journal. 58 (1): 5–13. doi:10.1002/aic.13701. ISSN 1547-5905.
- Stiege, Wolfgang; Erdmann, Volker A. (1995). "The potentials of the in vitro protein biosynthesis system". Journal of Biotechnology. 41 (2–3): 81–90. doi:10.1016/0168-1656(95)00005-b. PMID 7654353.
- Matthaei H.; Nirenberg (1962). "Characteristics and Stabilization of DNAase-Sensitive Protein Synthesis in E. coli Extracts". Proceedings of the National Academy of Sciences of the United States of America. 47 (10): 1580–1588. Bibcode:1961PNAS...47.1580M. doi:10.1073/pnas.47.10.1580. PMC 223177. PMID 14471391.
- Anderson, Carl W.; Straus, J.William; Dudock, Bernard S. (1983). " Preparation of a cell-free protein-synthesizing system from wheat germ". Recombinant DNA Part C. Methods in Enzymology. 101. pp. 635–644. doi:10.1016/0076-6879(83)01044-7. ISBN 9780121820015. PMID 6888279.
- Madin, Kairat; Sawasaki, Tatsuya; Ogasawara, Tomio; Endo, Yaeta (2000-01-18). "A highly efficient and robust cell-free protein synthesis system prepared from wheat embryos: Plants apparently contain a suicide system directed at ribosomes". Proceedings of the National Academy of Sciences. 97 (2): 559–564. Bibcode:2000PNAS...97..559M. doi:10.1073/pnas.97.2.559. ISSN 0027-8424. PMC 15369. PMID 10639118.
- Woodward, William R.; Ivey, Joel L.; Herbert, Edward (1974). "[67a] Protein synthesis with rabbit reticulocyte preparations". Nucleic Acids and Protein Synthesis Part F. Methods in Enzymology. 30. pp. 724–731. doi:10.1016/0076-6879(74)30069-9. ISBN 9780121818937. PMID 4853925.
- Y. H. Percival Zhang (March 2010). "Production of biocommodities and bioelectricity by cell-free synthetic enzymatic pathway biotransformations: Challenges and opportunities". Biotechnology and Bioengineering. 105 (4): 663–677. doi:10.1002/bit.22630. PMID 19998281.
- Mehta, Preeti; Woo, Perry; Venkataraman, Krithika; Karzai, A. Wali (2012). Bacterial Regulatory RNA. Methods in Molecular Biology. 905. Humana Press, Totowa, NJ. pp. 273–289. doi:10.1007/978-1-61779-949-5_18. ISBN 9781617799488. PMC 4607317. PMID 22736011.
- Zhang YH, Evans BR, Mielenz JR, Hopkins RC, Adams MW (2007). "High-Yield Hydrogen Production from Starch and Water by a Synthetic Enzymatic Pathway". PLoS ONE. 2 (5): e456. Bibcode:2007PLoSO...2..456Z. doi:10.1371/journal.pone.0000456. PMC 1866174. PMID 17520015.
- You C, Chen H, Myung S, Sathitsuksanoh N, Ma H, Zhang XZ, Li J, Zhang YH (2013). "Enzymatic transformation of nonfood biomass to starch". Proceedings of the National Academy of Sciences. 110 (18): 7182–7187. Bibcode:2013PNAS..110.7182Y. doi:10.1073/pnas.1302420110. PMC 3645547. PMID 23589840.
- Zhu Z, Kin Tam T, Sun F, You C, Percival Zhang YH (2014). "A high-energy-density sugar biobattery based on a synthetic enzymatic pathway". Nature Communications. 5: 3026. Bibcode:2014NatCo...5.3026Z. doi:10.1038/ncomms4026. PMID 24445859.
- Wang, Yiran; Huang, Weidong; Sathitsuksanoh, Noppadon; Zhu, Zhiguang; Zhang, Y.-H. Percival (2011). "Biohydrogenation from Biomass Sugar Mediated by In Vitro Synthetic Enzymatic Pathways". Chemistry. 18 (3): 372–380. doi:10.1016/j.chembiol.2010.12.019. PMID 21439482.
- Nirenberg, M.W. & Matthaei, H.J. (1961). "The Dependence Of Cell- Free Protein Synthesis In E. coli Upon Naturally Occurring Or Synthetic Polyribonucleotides". Proceedings of the National Academy of Sciences of the United States of America. 47 (10): 1588–1602. Bibcode:1961PNAS...47.1588N. doi:10.1073/pnas.47.10.1588. PMC 223178. PMID 14479932.
- Spirin, A. S.; Baranov, V. I.; Ryabova, L. A.; Ovodov, S. Y.; Alakhov, Y. B. (1988-11-25). "A continuous cell-free translation system capable of producing polypeptides in high yield". Science. 242 (4882): 1162–1164. Bibcode:1988Sci...242.1162S. doi:10.1126/science.3055301. ISSN 0036-8075. PMID 3055301.
- Yang, Junhao; Kanter, Gregory; Voloshin, Alexei; Michel-Reydellet, Nathalie; Velkeen, Hendrik; Levy, Ronald; Swartz, James R. (2005-03-05). "Rapid expression of vaccine proteins for B-cell lymphoma in a cell-free system". Biotechnology and Bioengineering. 89 (5): 503–511. doi:10.1002/bit.20283. ISSN 1097-0290. PMID 15669088.
- Bujara, Matthias; Schümperli, Michael; Pellaux, René; Heinemann, Matthias; Panke, Sven (2011). "Optimization of a blueprint for in vitro glycolysis by metabolic real-time analysis" (PDF). Nature Chemical Biology. 7 (5): 271–277. doi:10.1038/nchembio.541. PMID 21423171.
- Calhoun, Kara A.; Swartz, James R. (2005-06-05). "Energizing cell-free protein synthesis with glucose metabolism". Biotechnology and Bioengineering. 90 (5): 606–613. doi:10.1002/bit.20449. ISSN 1097-0290. PMID 15830344.
- Noren, C. J.; Anthony-Cahill, S. J.; Griffith, M. C.; Schultz, P. G. (1989-04-14). "A general method for site-specific incorporation of unnatural amino acids into proteins". Science. 244 (4901): 182–188. Bibcode:1989Sci...244..182N. doi:10.1126/science.2649980. ISSN 0036-8075. PMID 2649980.
- Kigawa, Takanori; Muto, Yutaka; Yokoyama, Shigeyuki (1995-09-01). "Cell-free synthesis and amino acid-selective stable isotope labeling of proteins for NMR analysis". Journal of Biomolecular NMR. 6 (2): 129–134. doi:10.1007/bf00211776. ISSN 0925-2738.