Single‐step purification and structural characterization of human interleukin‐6 produced in Esherichia coli From a T7 RNA polymerase expression vector

Abstract

Human interleukin-6 or B-cell stimulatory factor-2 is a cytokine involved in acute phase and immune response. Cloning of cDNA for human interleukin-6 in the pT7.7 expression plasmid under the control of a bacteriophage T7 RNA polymerase promoter system allows rapid production of the cytokine in Escherichiu coli. Upon cell induction with isopropyl thiogalactopyranoside, recombinant human interleukin-6 is overexpressed and forms insoluble inclusion bodies. Solubilization of the protein with 6 M guanidine hydrochloride and refolding in the presence of a reduction/oxidation system results in a quantitative recovery of recombinant human interleukin-6. This material is already 70% pure and can be further purified to homogeneity with a single passage over a weak anionic-exchange column. Eytended structural Characterization of the purified protein by electrospray mass spectrometry, automated Edman degradation and peptide mapping by high-pressure liquid chromatography/fastatom-bombardment mass spectrometry demonstrates that recombinant human interleukin-6 is identical to the natural protein both in amino acid sequence and S-S bridge content. However, it contains a minor component accounting for about 20% of the entire translated protein which exhibits a Met-Ala dipeptide extension at the Nterminus. Purified recombinant human interleukin-6 is biologically active because it is able to induce at least 70-fold the human C-reactive promoter transfected in human hepatoma Hep 3B cells and is stable for several months in 10% glycerol at 4°C. The expression system described in the present work has the main advantage of producing a high yield of recombinant human interleukin-6 (about 25 mg/l) combined with a very simple purification scheme.Interleukin-6 (IL-6) or B-cell stimulatory factor 2, is a major cytokine which has been shown to play a central role in the physiology of several tissues and organs [l]. The main targets of IL-6 are the immune system and the liver [l, 21. On B cells IL-6 induces the growth of hybridomas and plasmacytomas, the expression of class I antigens and the production and secretion of immunoglobulins [l]. On hepatocytes IL-6 is a transcriptional inducer of a large group of plasma proteins, including C-reactive protein, al-acid glycoprotein, haptoglobin, hemopexin, fibrinogen, etc., which are also known as acute phase proteins or reactants [2]. IL-6, however, is also involved in the differentiation of T-cells [3] and neurons [4]. Besides the physiological activities of IL-6, deregulation of the expression of this cytokine has been postulated to be responsible for the development of several

Interleukin-6 and oncostatin M-induced growth inhibition of human A375 melanoma cells is STAT-dependent and involves upregulation of the cyclin-dependent kinase inhibitor p27/Kip1

“…Cells were grown in RPMI 1640 medium supplemented with 5% FCS, 50 mg/ ml penicillin, 100 mg/ml streptomycin in a humidi®ed atmosphere with 5% CO 2 . Puri®ed recombinant human IL-6 with a speci®c activity of 2610 6 B cell stimulatory factor-2 units/mg was prepared as described (Arcone et al, 1991). Soluble human IL-6 receptor was expressed in baculovirusinfected insect cells (WeiergraÈ ber et al, 1995).…”

Hepatic cytochrome P450 down-regulation during aseptic inflammation in the mouse is interleukin 6 dependent

“…Recombinant human IL-6, with a specific activity of 5 ϫ 10 6 B-cell stimulatory factor 2 units/mg protein, was produced in the Escherichia coli expression system, purified to homogeneity by gel filtration and ion exchange high-performance liquid chromatography as described. 28 Endotoxin content was less than 3.5 pg/mg as measured in the Limulus assay, which is far below the concentration of LPS needed to induce an acute phase protein production. 25 Animals and Treatments.…”

Purification and Characterization of the Soluble Interleukin‐6 Receptor from Human Plasma and Identification of An Isoform Generated through Alternative Splicing

“…For the preparation of the affinity columns 2.5 mg mAb5 or 2.5 mg recombinant hIL-6, which was expressed in Escherichia coli and purified from inclusion bodies as described [26], were immobilized to 285 mg cyanogen-bromide-activated Sepharose (Pharmacia) according to the instructions of the manufacturer. Frozen donor plasma was kindly provided by the blood bank of the Klinikum Aachen; 3 ml 1 M CaC1, was added to 300 ml plasma and, after incubation at 37°C for 90 min, 10 ml 0.5 M EDTA was added.…”