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 Escherichia coli. The 163‐residue protein des‐(A1–S22)‐[C45S, CS1S]interleukin‐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 interleukin‐6 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 ± 0.4 kJ · mol−1 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. Copyright © 1995, Wiley Blackwell. All rights reserved