Journal of Chromatography B, 853 (2007) 314–319 Two-step method to isolate target recombinant protein from co-purified bacterial contaminant SlyD after immobilised metal affinity chromatography C´ eline B. Parsy a,∗ , Caroline J. Chapman b , Antony C. Barnes a , John F. Robertson b , Andrea Murray a a OncImmune Limited, Clinical Sciences Building, Nottingham City Hospital, Hucknall Road, Nottingham NG5 1PB, UK b Tumor Immunology Group, University of Nottingham, Division of Breast Surgery, Nottingham City Hospital, Hucknall Road, Nottingham NG5 1PB, UK Received 26 January 2007; accepted 25 March 2007 Available online 8 April 2007 Abstract As part of a study to purify the internal domain of HER2 (ICD) from recombinant expression, through metal immobilised affinity chromatography (IMAC), we encountered a contaminant, SlyD, a 29 kDa native E. coli protein. SlyD is a recurrent contaminant, with a histidine rich domain enabling binding to IMAC columns and thus co-elution with the target protein. Research has been carried out on this protein and its purification, however, no work mentions how to treat it as a true contaminant or describe procedures to isolate it from target proteins. In this report, we described a two-step chromatographic method for the purification of ICD, including IMAC as a capture step and size exclusion chromatography (SEC) as a polishing step. IMAC allowed us to purify ICD from bacterial crude with SlyD co-eluting. SEC then allowed us to resolve ICD from SlyD and achieve a purity greater than 95% for ICD. However, this method has been developed to accommodate any protein whose molecular weight is different enough from SlyD to be separated by SEC. © 2007 Elsevier B.V. All rights reserved. Keywords: Recombinant protein; SlyD; Size exclusion chromatography; Immobilized metal affinity chromatography 1. Introduction The past 20 years have seen the development of techniques and tools for the expression and purification of recombi- nant proteins which have transformed the way scientists approach protein production and research. Vector constructs allow over-expression of an increasing amount of protein- encoding DNA sequences. Naturally low-abundant protein can then be expressed in gram quantity through a suitable expres- sion vector and host combination. Some vectors can also insert extra DNA sequences leading to the production of tagged proteins, which incorporate extra amino-acid sequences like hexa-histidine [1], GST [2] or streptavidin [3]. These tags can then be used to purify the recombinant protein by single-step affinity chromatography through corresponding matrix and lig- ∗ Corresponding author. Tel.: +44 115 823 1871; fax: +44 115 823 1881. E-mail address: celine.parsy@nottingham.ac.uk (C.B. Parsy). and [4,5]. Immobilised metal affinity chromatography (IMAC) is one such example and generally involves the fusion of a six- histidine tag to the target protein, though the tag can vary in length (4–10 histidines). This tag interacts with a chromatogra- phy matrix charged with metal ions (mainly nickel and cobalt) due to the affinity of histidine for these ions. As a result, recom- binant fusion protein can be purified to homogeneity from a bacterial lysate in only one step. However, the progress in recombinant protein expression and purification has raised new challenges. The over-expression of a target protein by the host bacteria has been linked to the over- expression of native bacterial proteins, as a response to stress conditions following the high production of foreign material [6–8]. Some of these native proteins have properties that cause them to co-purify with the target protein when IMAC is being used, as they show an affinity to divalent cations [9]. The most studied recurrent contaminant of IMAC purification is SlyD, a 196 amino acids protein, known to be present in E. coli dur- ing lysis when infected by bacteriophage X174 [10]. SlyD 1570-0232/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2007.03.046