title: Protein production for structural genomics using E. coli expression.
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.
authors.affiliation: Center for Structural Genomics of Infectious Diseases, Computational Institute, University of Chicago, Chicago, IL, 60557, USA.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Makowska-Grzyska
authors.forename: Youngchang
authors.initials: Y
authors.lastname: Kim
authors.forename: Natalia
authors.initials: N
authors.lastname: Maltseva
authors.forename: Hui
authors.initials: H
authors.lastname: Li
authors.forename: Min
authors.initials: M
authors.lastname: Zhou
authors.forename: Grazyna
authors.initials: G
authors.lastname: Joachimiak
authors.forename: Gyorgy
authors.initials: G
authors.lastname: Babnigg
authors.forename: Andrzej
authors.initials: A
authors.lastname: Joachimiak
mesh_terms: D019295:Computational Biology; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D008967:Molecular Biology / Q000379:methods*; D011506:Proteins / Q000737:chemistry / Q000235:genetics*; D040901:Proteomics / Q000379:methods; D059467:Transcriptome
chemical_list: D011506:Proteins