Journal of Chromatography A, 1216 (2009) 4451–4456 Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma Efficient purification of recombinant proteins fused to maltose-binding protein by mixed-mode chromatography Charlotte Cabanne ∗ , Jérôme Pezzini, Gilles Joucla, Agnès Hocquellet, Caroline Barbot, Bertrand Garbay, Xavier Santarelli EA 4135, ESTBB, Université V. Segalen Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux cedex, France article info Article history: Received 6 January 2009 Received in revised form 10 March 2009 Accepted 16 March 2009 Available online 20 March 2009 Keywords: Mixed-mode chromatography Maltose-binding protein Recombinant protein purification abstract Two mixed-mode resins were evaluated as an alternative to conventional affinity resins for the purifi- cation of recombinant proteins fused to maltose-binding protein (MPB). We purified recombinant MBP, MBP-LacZ and MBP-Leap2 from crude Escherichia coli extracts. Mixed-mode resins allowed the efficient purification of MBP-fused proteins. Indeed, the quantity of purified proteins was significantly higher with mixed-mode resins, and their purity was equivalent to that obtained with affinity resins. By using purified MBP, MBP-LacZ and MBP-Leap2, the dynamic binding capacity of mixed-mode resins was 5-fold higher than that of affinity resins. Moreover, the recovery for the three proteins studied was in the 50–60% range for affinity resins, and in the 80–85% range for mixed-mode resins. Mixed-mode resins thus represent a powerful alternative to the classical amylose or dextrin resins for the purification of recombinant proteins fused to maltose-binding protein. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Expression of recombinant proteins in heterologous organisms usually requires fusion of the said protein with a polypeptide part- ner. This fusion partner, or tag, facilitates the purification process and in several cases the detection of the recombinant protein. More- over, it may improve the solubility of the recombinant protein, thereby preventing the formation of biologically inactive aggre- gates. Maltose-binding protein (MBP) has been widely used as a fusion partner for the production of recombinant protein in Escherichia coli [1]. It allows a high level of expression, increases the solubility of the fused protein, and binds specifically to an immobilized ligand composed of amylose or dextrin, thus facil- itating the purification process [2–5]. Nevertheless, purification of MBP-fusion proteins by affinity chromatography may be less straightforward than usually expected. Hence, it is thought that the affinity of MBP for amylose or dextrin resins depends on multiple factors. The nature of the protein fused to the MBP can influence the binding capacity of MBP, which may lead to lower purification yields [6]. In one case, it was demonstrated that the fusion partner interacted with the maltodextrin-binding site of MBP, thus prevent- ing the correct binding of the fusion protein during the amylose chromatography step [7]. Moreover, endogenous -amylases pro- duced by E. coli can degrade the Amylose resin, and thus decrease ∗ Corresponding author. E-mail address: charlotte.cabanne@estbb.u-bordeaux2.fr (C. Cabanne). the purification yield [8]. To circumvent these problems, several authors added a hexahistidine tag (His6) to their MBP-fusion con- structs [6,9]. Their goal was to keep the solubility effect mediated by MBP, and to use immobilized metal affinity chromatography (IMAC) to purify the recombinant protein throughout its His-tag. Further- more, a one-step chromatography process on IMAC was successfully used to purify a recombinant protein fused to MBP, suggesting that MBP itself can interact with copper-chelating columns [10]. MBP has an unusually large number of exposed hydrophobic zones on its surface [3]. Based on this observation, we tested an alternative mixed-mode chromatography method to purify MBP- fused proteins. We used two recently developed mixed-mode resins: HEA and PPA HyperCel [11]. Using these resins, protein purification is based on hydrophobic, ionic or electrostatic and/or pseudo-affinity interactions during the binding step and essentially ionic interactions (charge repulsion) during the elution step. This dual mode technique based on ionisable ligands was first termed hydrophobic charge induction chromatography [12]. It is aimed at the purification of proteins in low-to-moderate salt concentra- tions when compared to the traditional hydrophobic interaction chromatography (HIC). The possibility to bind proteins under neu- tral “physiological-like” conditions minimizes their precipitation and/or aggregation, and may contribute to a better recovery. Mixed- mode chromatography has been used as an alternative to Protein A or HIC chromatography to purify monoclonal antibodies [12–18], and to purify other proteins such as serum albumin or penicillin acylase [19–23]. In this study, we investigated the potential of HEA and PPA HyperCel resins to purify MBP and two recombinant MBP- 0021-9673/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2009.03.048