Matrix-assisted refolding and purification of placenta-derived recombinant human interleukin-6 produced in Escherichia coli Nadeem Ahmed ∗ Ahmad Usman Zafar Mohsin Ahmad Khan Saad Tahir Muhammad Islam Khan Hamid Bashir Faidad Khan Samreen Sarwar Sadaf Ilyas Tayyab Husnain National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan Abstract Biological activity of human interleukin-6 (IL-6) is associated with a vast number of diseases such as rheumatoid arthritis, sepsis, and severe inflammatory diseases. In this study, human IL-6 cDNA was isolated from a cDNA library that was constructed with mRNA derived from human placental tissues. Subsequently, the complete human IL-6 cDNA was cloned and expressed in BL21DE3 cells. The recombinant human IL-6 (rhIL-6) protein was expressed in a form of an insoluble inclusion body. Inclusion bodies were solubilized under denaturing conditions and purified by immobilized metal affinity chromatography with gradual on-column refolding by the gradient elution method (from 6 to 0 M urea). The protein was purified to apparent homogeneity of about 99% with a yield of 50 mg/L. The purity was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), size exclusion high-performance liquid chromatography, and Western blotting analysis. The bioactivity was assessed by proliferation assay of TF-1 cells in a dose-dependent manner. The present study confirms the expression of the placenta-derived IL-6 gene in a prokaryotic expression system and matrix-assisted on-column refolding and purification of rhIL-6 by immobilized metal affinity chromatography. C  2014 International Union of Biochemistry and Molecular Biology, Inc. Volume 61, Number 5, Pages 541–548, 2014 Keywords: immobilized metal-ion-affinity chromatography, pET28 expression vector, on-column refolding, chelating sepharose fast flow, histidine tags, recombinant proteins 1. Introduction Interleukin-6 (IL-6), a single-chain glycoprotein, executes signals for induction of differentiation of B cells and differential Abbreviations: IL-6, interleukin-6; IMAC, immobilized metal-ion-affinity chromatography; Ni-Sepharose, Ni 2+ sepharose; rhIL-6, recombinant human interleukin-6; LB, Luria–Bertani. ∗ Address for correspondence: Nadeem Ahmed, National Centre of Excellence in Molecular Biology, University of the Punjab, 87 West Canal Bank Road, Lahore 53700, Pakistan. Tel.:+9204235293141; Fax:+9204235293147; e-mail: nadeem.ahmed@cemb.edu.pk. Received 29 July 2013; accepted 27 December 2013 DOI: 10.1002/bab.1205 Published online 1 April 2014 in Wiley Online Library (wileyonlinelibrary.com) stimulation or inhibition of cell growth and has different cellular sources, including immune cells, various cell lines, and tumor cells. In different tissues, different mechanisms regulate the expression of IL-6 owing to the presence of multiple initiation sites [1]. In addition, IL-6 with its functional pleiotropy and redundancy in action, is involved in pathophysiology of various diseases, e.g., multiple myeloma, rheumatoid arthritis, and osteoporosis [2–4]. Its role in an acute phase reaction has also been well established [5–7]. IL-6 was found to be a reliable treatment for diseases in cases where standard antibiotic-based strategies have failed [8–10]. In addition, IL6 is a potent clinical target for cancer therapy. IL-6 inhibits the progression of solid tumors such as mammary carcinomas, cervical carcinomas, human lung cancers, histolytic lymphomas, and melanomas [11, 12]. Therefore, it is required that a reliable method for preparation 541