Stefan B. Schmidbauer and Oliver K. Strobel

Boehringer Mannheim GmbH, Penzberg, Germany

2. INTRODUCTION

Metal chelate or immobilized metal affinity chromatography (IMAC) is a powerful technique for isolating recombinant fusion proteins under mild conditions (1, 2). A metal chelating group is first immobilized on a chromatographic medium, and a multivalent metal ion (usually Cu²⁺, Ni²⁺, Zn²⁺ or Co²⁺) is bound in a way that leaves some coordination sites free for selective interaction with proteins. Typically, 5-6 histidine residues ("tag") are added to the C- or N-terminus of a target protein using recombinant techniques. The tag specifically interacts with the chelated metal ions, thereby holding these proteins on the medium. Other components bind weakly or not at all. Elution of the fusion protein is usually performed by increasing the concentration of a competitive eluting agent, such as imidazole, or by reducing the pH. POROS chromatography media have a unique structure that allows separation of biomolecules to be carried out significantly faster than on conventional media. Resolution and capacity are maintained at these high flow rates and the technique is known as Perfusion Chromatography (3). This article describes the rapid one-step purification of a histidine-tagged recombinant fusion protein, glutathione 5-transferase, GST(His)₆, from bacterial lysate with POROS MC (Metal Chelate) media.