Review Structural analysis and classification of native proteins from E. coli commonly co-purified by immobilised metal affinity chromatography Victor Martin Bolanos-Garcia ⁎ , Owen Richard Davies Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, England Received 25 January 2006; received in revised form 23 March 2006; accepted 24 March 2006 Available online 26 April 2006 Abstract Immobilised metal affinity chromatography (IMAC) is the most widely used technique for single-step purification of recombinant proteins. However, despite its use in the purification of heterologue proteins in the eubacteria Escherichia coli for decades, the presence of native E. coli proteins that exhibit a high affinity for divalent cations such as nickel, cobalt or copper has remained problematic. This is of particular relevance when recombinant molecules are not expressed at high levels or when their overexpression induces that of native bacterial proteins due to pleiotropism and/or in response to stress conditions. Identification of such contaminating proteins is clearly relevant to those involved in the purification of histidine-tagged proteins either at small/medium scale or in high-throughput processes. The work presented here reviews the native proteins from E. coli most commonly co-purified by IMAC, including Fur, Crp, ArgE, SlyD, GlmS, GlgA, ODO1, ODO2, YadF and YfbG. The binding of these proteins to metal-chelating resins can mostly be explained by their native metal-binding functions or their possession of surface clusters of histidine residues. However, some proteins fall outside these categories, implying that a further class of interactions may account for their ability to co-purify with histidine-tagged proteins. We propose a classification of these E. coli native proteins based on their physicochemical, structural and functional properties. © 2006 Elsevier B.V. All rights reserved. Keywords: Affinity chromatography; E. coli contaminant protein; Metal-binding classification; Histidine-tagged protein; Protein purification 1. Introduction The use of immobilised metal affinity chromatography (IMAC) has revolutionised protein biochemistry by allowing the production of a pure protein sample through a single puri- fication step. However, the concomitant expression of native bacterial proteins that exhibit a relatively high affinity for di- valent cations during the expression of heterologue protein domains, full-length proteins or macromolecular complexes in E. coli frequently results in their co-purification during IMAC [1]. Most of these metal binding proteins are present in E. coli strains of different genetic backgrounds, such as BL21, BL21 (DE3), BL21 (DE3) pLysS, C41, C43, Rosetta (DE3) and (DE3) pLysS as well as Origami (DE3) and (DE3) pLysS. These strains contain a lamba-lysogen DE3 bacteriophage that encodes T7 RNA polymerase under the control of the lac UV5 operator; the expression of T7 promoter and lac UV5 operator controlled genes on pET-based vectors is thus permitted upon induction with isopropyl-2-D-thio-galactopyranoside (IPTG) [2,3]. Since the 1970s, IMAC has remained the most important technique for single-step protein purification [4]. The expression of a recombinant protein containing a histidine-tag (usually six consecutive histidine residues) allows it to be specifically bound by chelated divalent metal ions, and then eluted through com- petition by the addition of imidazole, or through the protonation of histidine residues by a reduction in pH. This often has dramatic results in the purification of target proteins to near homogeneity from bacterial cell lysate [5,6]. Further advantages of IMAC include ligand stability, high protein loading capacity, mild or denaturing elution conditions, column regeneration, low cost and scalability [7,8]. This has meant that it is now in widespread use in both low and high throughput environments [9,10]. There are many different metal-chelator systems for IMAC, although the most common are the tridentate ligand IDA, Ni 2+ bound to tetradentate ligand NTA (Ni-NTA; Qiagen Ltd.) and Co 2+ Biochimica et Biophysica Acta 1760 (2006) 1304 – 1313 www.elsevier.com/locate/bbagen ⁎ Corresponding author. Tel.: +44 1223 766029; fax: +44 1223 766002. E-mail address: victor@cryst.bioc.cam.ac.uk (V.M. Bolanos-Garcia). 0304-4165/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2006.03.027