Overview of the Purification of Recombinant Proteins Produced inEscherichia coli

Abstract

The updated version of this unit presents an overview of recombinant protein purification with special emphasis on proteins expressed in E. coli. The first section deals with information pertinent to protein purification that can be derived from translation of the cDNA sequence. This is followed by a discussion of common problems associated with bacterial protein expression. A flow chart summarizes approaches for establishing solubility and localization of bacterially produced proteins. Purification strategies for both soluble and insoluble proteins are also reviewed. A section on glycoproteins produced in bacteria in the nonglycosylated state is included to emphasize that, although they may not be useful for in vivo studies, such proteins are well suited for structural studies. Finally, protein handling, scale and aims of purification, and specialized equipment needed for recombinant protein purification and characterization are discussed. The methodologies and approaches described here are essentially suitable for laboratory-scale operations.

Anti-human fibroblast growth factor-21 monoclonal antibody preparation, characterization and analysis of in vitro bioactivity

“…Different hFGF-21 segments were anchored to the inner membrane of the host cell, in order to allow screening for the epitope that anti-hFGF-21 mAb (2D8) recognizes. Bacterial cells are the host of choice for expression of recombinant proteins, due to their fast expression foreign genes, rapid growth rate and ease of genetic manipulation (20,(22)(23)(24)(25)(26). APEx-FACS enables real-time visualization of mAb-peptide binding, which allowed identification of the epitope that the anti-hFGF-21 mAb recognized.…”

Analysis of Proteins

“…10.0.4; also see Wingfield, 2002Wingfield, , 2005. To carry out a purification it is nearly always necessary to obtain the desired protein in a soluble form, which will often require the addition of solubilizing agents such as detergents.…”

So you Need a Protein - A Guide to the Production of Recombinant Proteins

“…The first choice of an expression system for the production of recombinant proteins for many investigators is E. coli [8]. The E. coli expression system has many advantages compared with other expression systems such as easy growth conditions, rapid biomass accumulation, and a simple scaleup process [9].…”

“…Clones expressing proteins can be identified by SDS-PAGE and Western blotting with antisera, either directed at the recombinant itself, or the tag (typically a monoclonal antibody that recognises the His-tag or another tag). An expressing clone is identified and used in 50-250 mL culture volumes for further optimisation, refinement of expression conditions and optimisation of purification [8].…”

“…If the protein aggregates into inclusion bodies, approximately 100 to 300 mg of recombinant protein is available (2% to 5% of total proteins) [7,8,10]. If the protein is soluble (usually desirable) yields will be somewhat less.…”