Abstract
The goal of structural biology is to reveal details of the molecular structure of proteins in order to understand their function and mechanism. X-ray crystallography and NMR are the two best methods for atomic level structure determination. However, these methods require milligram quantities of proteins. In this chapter a reproducible methodology for large-scale protein production applicable to a diverse set of proteins is described. The approach is based on protein expression in E. coli as a fusion with a cleavable affinity tag that was tested on over 20,000 proteins. Specifically, a protocol for fermentation of large quantities of native proteins in disposable culture vessels is presented. A modified protocol that allows for the production of selenium-labeled proteins in defined media is also offered. Finally, a method for the purification of His6-tagged proteins on immobilized metal affinity chromatography columns that generates high-purity material is described in detail.
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Acknowledgments
This work was supported by the National Institute of Allergy and Infectious Diseases Contract (HHSN272200700058C and HHSN272201200026C), the National Institute of Health Grant GM094585, and the US Department of Energy office of Biological and Environmental Research under Contract No. DE-AC02-06CH11357. This work has been created by UChicago Argonne, LLC, Operator of Argonne National Laboratory (“Argonne”). Argonne, a US Department of Energy Office of Science laboratory, is operated under Contract No. DE-AC02-06CH11357. The US Government retains for itself, and others acting on its behalf, a paid-up nonexclusive, irrevocable worldwide license in said article to reproduce, prepare derivative works, distribute copies to the public, and perform publicly and display publicly, by or behalf of the government.
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Makowska-Grzyska, M. et al. (2014). Protein Production for Structural Genomics Using E. coli Expression. In: Anderson, W.F. (eds) Structural Genomics and Drug Discovery. Methods in Molecular Biology, vol 1140. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-0354-2_7
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DOI: https://doi.org/10.1007/978-1-4939-0354-2_7
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