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Methods Mol Biol. 2014;1140:89-105. doi: 10.1007/978-1-4939-0354-2_7.

Protein production for structural genomics using E. coli expression.

Author information

1
Center for Structural Genomics of Infectious Diseases, Computational Institute, University of Chicago, Chicago, IL, 60557, USA.

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.

PMID:
24590711
PMCID:
PMC4108990
DOI:
10.1007/978-1-4939-0354-2_7
[Indexed for MEDLINE]
Free PMC Article

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