Purification and characterization of a recombinant murine interleukin‐6: Isolation of N‐ and C‐terminally truncated forms

Murine interleukin‐6 (IL‐6), when expressed in Escherichia coli using the pUC9 vector, accumulated as insoluble aggregates or ‘inclusion bodies’. After selective urea washing of the inclusion bodies, to remove extraneous proteins, murine IL‐6 was solubilized with 8 M guanidine hydrochloride and then rapidly purified to homogeneity by gel‐permeation chromatography followed by reversed‐phase HPLC. It was demonstrated that complete disulfide bond formation in murine IL‐6 occurred during the early urea washing/guanidine hydrochloride extraction steps, so no refolding step was required. When fully reduced murine IL‐6 was dissolved in 8 M guanidine hydrochloride and allowed to airoxidize, complete disulfide bond formation, monitored by analytical reversed‐phase HPLC, was shown to occur within 13 h at 6°C. About 25 mg pure protein was obtained from 37 g wet cells. This recombinant murine IL‐6 had a specific activity in the hybridoma growth factor assay of 2×10⁸ U/mg, which is equivalent to that of native murine IL‐6. During the purification procedure, a number of variant forms of murine IL‐6 were isolated and partially characterized. Two of these forms, T1 and T3, were C‐terminal deletants of murine IL‐6 lacking about 60 and 20 amino acids from the C‐terminus, respectively, while the other form, T2, was an N‐terminal deletant lacking 37 amino acids from the N‐terminus. None of these variant forms of murine IL‐6 bound to the murine IL‐6 receptor and, consequently, all were inactive in the hybridoma growth factor assay.