Summary
One of the main challenges in this post genomic era is the development and implementation of efficient methods of protein synthesis. A clear understanding of the role of genes in an organism is to comprehend the biological functions of all of its proteins. Acquiring this knowledge will depend in part on the success of rapid synthesis and purification of proteins. The future of structural genomics and functional proteomics depends on the availability of abundantly expressing, soluble proteins in a high-throughput manner. Conventional cell based methods of protein expression is rather laborious, time consuming and the ways to fail are numerous including solubility, toxicity to the host and instability (e.g. proteolysis). Cell-free or in vitro protein synthesis, on the other hand allows the expression and analysis of protein synthesis, may solve many of these problems. It is a simple open system which lends itself for manipulations and modifications to influence protein folding, disulfide bond formation, incorporation of unnatural amino acids, protein stability (by incorporating protease inhibitors in the system) and even the expression of toxic proteins. Cell-free synthesis can also be used as a reliable screening methodology for subsequent protein expression in vivo. Furthermore, this technology is readily amenable to automation. Here, we present a protocol for expressing recombinant proteins with high yield in a standard 96-well plate format using E. coli cell-free extract in a batch mode.
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Acknowledgments
This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract N01-CO-12400. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Sitaraman, K., Chatterjee, D.K. (2009). High-Throughput Protein Expression Using Cell-Free System. In: Doyle, S.A. (eds) High Throughput Protein Expression and Purification. Methods in Molecular Biology, vol 498. Humana Press. https://doi.org/10.1007/978-1-59745-196-3_15
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DOI: https://doi.org/10.1007/978-1-59745-196-3_15
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