Biotechnol. Appl. Biochem. (2010) 55, 209–214 (Printed in Great Britain) doi:10.1042/BA20090256 209 Purification and refolding of Escherichia coli-expressed recombinant human interleukin-2 Samaneh Esfandiar*, Sameereh Hashemi-Najafabadi* 1 , Seyed Abbas Shojaosadati*, Shokuh Aazam Sarrafzadeh† and Zahra Pourpak† *Biotechnology Group, Chemical Engineering Department, Tarbiat Modares University, P.O. Box 14115-143, Tehran, Iran, and †Immunology, Asthma and Allergy Research Institute, Tehran University of Medical Sciences, P. O. Box 14185-863, Tehran, Iran The expression of rhIL-2 (recombinant human interleukin-2) in bacteria results in the formation of insoluble inclusion-body aggregates. These aggregates were first solubilized under denaturing conditions (sodium phosphate buffer solution containing 8M urea and 10 mM 2-mercaptoethanol) and then purified using IMAC (immobilized metal-ion-affinity chromato- graphy). IMAC was used to capture rhIL-2. The protein was gradually refolded on the column by a gradient elution (8 M to 0 M urea) in the presence of 10% (v/v) glycerol. Glycerol was used to prevent protein aggregation during the refolding step. Using this method, rhIL-2 was collected at 97 % purity and its activity was measured by the lymphocyte transformation test. The measured activity was identical with commercial human interleukin-2. Introduction IL-2 (interleukin-2), an important cytokine, is synthesized and produced by lymphocytes. This 133-amino-acid protein has three cysteine residues at positions 58, 105 and 125 [1,2]. Its direct effects on immunological cells are apparent. It promotes the proliferation of activated T-cells, stimulates their growth and enables the differentiation and activation of T-, B- and natural killer cells. In recent years, rhIL-2 (recombinant human IL-2) has gained significant attention as a therapeutic agent for certain cancers. Its safe use for im- munotherapy has been recognized. Clinical trials have shown that IL-2-based therapy is promising for chronic infectious diseases, including HIV infection and hepatitis C [3–6]. IMAC (immobilized metal-ion-affinity chromatography) has been described as an efficient purification method for proteins from recombinant micro-organisms [7,8]. IMAC has the potential to become one of the most widely used chromatographic techniques because of its low cost, simplicity, and ability to work under both native and denaturing conditions. This ability allows for subsequent on-column renaturation. The high affinity and specificity of this method results in highly efficient purification [9]. In spite of its advantages, IMAC has not gained popularity due to concerns regarding leaching of metal ions and reproducibility. These concerns need to be evaluated prior to large-scale use of the process [9,10]. It should be noted that nickel concentrations observed in tetradentate IMAC-protein preparations are below the typical daily intake of nickel [10]. Qiagen has presented results that demonstrate that Ni-NTA (Ni 2+ -nitrilotriacetate)–agarose and Ni-NTA Superflow, under native and denaturing conditions, reproducibly produce proteins that have high stability and low levels of metal and ligand leaching. This result shows that Ni-NTA–agarose allows chromatographic purification of His 6 -tagged proteins with high reproducibility, including both a low lot-to-lot variability and high run- to-run reproducibility. Ni-NTA matrices provide protein elution fractions that are not contaminated with metal ions. These eluents are not affected by long-term storage in a harsh denaturant such as 0.1 M NaOH (the recommended concentration) or after repeated use of a packed chromatography column [11]. However, it should be mentioned that, in the case of pharmaceutical-grade proteins, His-tags should be removed after the IMAC step. Chemical cleavage is usually avoided; instead, proteolytic enzymes are used [10]. Various tags have been developed to simplify the purification process. Polypeptides, small proteins and enzymes added to the N- or C-terminus of the recombinant protein have been used as fusion tags. The biochemical features of each tag influence the stability, solubility and expressivity of the attached proteins [12,13]. The most commonly used tag for purification and detection of recombinantly expressed proteins is the poly- histidine tag His 6 . Protein purification using His 6 relies on Key words: His 6 -tagged recombinant human interleukin-2 (rhIL-2), immobilized metal-ion-affinity chromatography (IMAC), lymphocyte transformation test (LTT), metal-affinity chromatography purification, Ni 2+ -nitrilotriacetate–agarose (Ni-NTA–agarose), refolding. Abbreviations used: BrdU, bromodeoxyuridine; GPC, gel-permeation chromatography; IL-2, interleukin-2; IMAC, immobilized metal-ion-affinity chromatography; LB, Luria–Bertani; Ni-NTA, Ni 2+ -nitrilotriacetate; rhIL-2, recombinant human IL-2. 1 To whom correspondence should be addressed (email s.hashemi@modares.ac.ir). C  2010 Portland Press Limited