8 - System for High-Level Production in Escherichia coli and Rapid Purification of Recombinant Proteins: Application to Epitope Mapping, Preparation of Antibodies, and Structure—Function Analysis

Abstract

This chapter presents an application of the system for high-level production in Escherichia coli and rapid purification of recombinant proteins. The advent of gene cloning, the engineering of vectors for efficient expression, and the application of fast and high-flux methods for protein purification made available many recombinant proteins of biological interest. This represented a breakthrough for the structure–function analysis of bioactive proteins and cell receptors and facilitated X-ray crystallographic studies for definition of the three-dimensional structures of these proteins. The E. coli expression system allows the high-level production of recombinant proteins in authentic form, as fusion proteins with the [His]6 affinity tail and with mouse DHFR and the [His]6 tail. Because of the presence of the affinity tail, proteins that are produced in a soluble form or can be solubilized with GuHCl or urea can be purified almost to homogeneity in one step by nickel chelate affinity chromatography. The purified recombinant proteins simplify the production of monoclonal and polyclonal antibodies directed against defined regions of the native proteins.