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 affi nity tag that was tested on over 20,000 proteins. Specifi cally, a protocol for fermentation of large quantities of native proteins in disposable culture vessels is presented. A modifi ed protocol that allows for the production of selenium-labeled proteins in defi ned media is also offered. Finally, a method for the purifi cation of His 6 -tagged proteins on immobilized metal affi nity chromatography columns that generates highpurity material is described in detail.