Structure, Stability and Biological Properties of a N‐terminally Truncated form of Recombinant Human Interleukin‐6 Containing a Single Disulfide Bond

Abstract

A mutant species of the 185-residue chain of human interleukin-6 lacking 22-residues at its N-terminus and with a Cys+Ser substitution at positions 45 and 51 was produced in Escherichiu coli. The 163-residue protein des-(A1 -S22)-[C45S, CSISlinterleukin-6, containing a single disulfide bridge, formed inclusion bodies. Mutant interleukin-6 was solubilized in 6 M guanidine hydrochloride, subjected to oxidative refolding and purified to homogeneity by ammonium sulfate precipitation and hydrophobic chromatography. The purity of the mutant species was established by electrophoresis, isoelectrofocusing and reverse-phase HPLC and its structural identity was checked by N-terminal sequencing of both the intact protein and several of its proteolytic fragments. Electrospray mass spectrometry analysis of mutant interleukin-6 gave a molecular mass of 18 695 ? 2 Da in excellent agreement with the calculated value. Circular dichroic, fluorescence emission and second-derivative ultraviolet absorption spectra indicated that mutant interleukind maintains the overall secondary and tertiary structure, as well as stability characteristics, of the recombinant wild-type human interleukin-6. The urea-induced unfolding of mutant interleukin-6, monitored by circular dichroic measurements in the far-ultraviolet region, occurs as a highly cooperative process with a midpoint of denaturation at 5.5 M urea. The data of the reversible unfolding of mutant interleukin-6 mediated by urea were used to calculate a value of 20.9 2 0.4 kJ . mol-' for the thermodynamic stability of the protein at 25°C in the absence of denaturant. The biological activity of mutant interleukin-6 was evaluated in vitro by the hybridoma proliferation assay, and in vivo by measuring thrombopoiesis in monkeys. Dose/response effects of the mutant were comparable or even higher than those of the wild-type protein. Overall the results of this study show that mutant interleukin-6 is a biologically active cytokine, which could find practical use as a therapeutic agent.

Smad Proteins Suppress CCAAT/Enhancer-binding Protein (C/EBP) β- and STAT3-mediated Transcriptional Activation of the Haptoglobin Promoter

“…Reagents-Recombinant, N-terminally truncated, human IL-6 (mutein) (29) was kindly provided by Pharmacia Biocenter (Nerviano, Italy) and was used at 100 units/ml. Recombinant activin A was obtained from the NIDDK, National Institutes of Health, National Hormone and Pituitary Program and was used at 300 units/ml unless indicated otherwise.…”

The Plasmacytoma Growth Inhibitor Restrictin-P Is an Antagonist of Interleukin 6 and Interleukin 11

“…Hamster antimurine interferon ␥ antibodies were obtained from Genzyme Corp. IL-3 was purchased from Peprotec (Rocky Hill, NJ). Recombinant human and monoclonal rat anti-mouse IL-6 neutralizing antibodies were purchased from Genzyme Corp. Recombinant, N-terminally truncated, human IL-6 (mutein) (31) and basic fibroblast growth factor were kindly provided by Pharmacia Biocenter (Nerviano, Italy). Crude concentrated murine IL-6 was kindly provided by Dr. J. Lotem, Weizmann Institute, and murine IL-6 was obtained from Dr. J.…”

Interleukin‐6: Structure‐function relationships

“…At pH 4.0, where unfolding induced by urea follows a two-state process, recombinant wild-type mIL-6 has a thermostability of 9.0 kcal/mol (Ward et al, 1995b) compared with 14.5 kcal/mol for the related recombinant human growth hormone (hGH) (Brems et al, 1990). A variant of human IL-6, lacking the 22 N-terminal amino acids and the disulfide bond between Cys 44 and Cys 50 (these residues being substituted for serine), was shown to retain the properties of the full-length protein (Breton et al, 1995) and have a stable, protease resistant, molten globule-like conformation at pH 2.0 (de Filippis et al, 1996). The unfolding of this A-state follows a non two-state process with highly stable secondary structure (de Filippis et al, 1996).…”