PURIFICATION AND CHARACTERIZATION OF A RECOMBINANT MURINE INTERLEUKIN-6 - ISOLATION OF N-TERMINALLY AND C-TERMINALLY TRUNCATED FORMS

Zhang, JG; Moritz, RL; Reid, GE; Ward, LD; Simpson, RJ

Author(s): Zhang, JG; Moritz, RL; Reid, GE; Ward, LD; Simpson, RJ;
Details: Publication Year 1992-08-01,Volume 207,Issue #3,Page 903-913
Journal Title: EUROPEAN JOURNAL OF BIOCHEMISTRY
Publication Type: Journal Article
Abstract: 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 air-oxidize, complete disulfide bond formation, monitored by analytical reversed-phase HPLC, was shown to occur within 13 h at 6-degrees-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 x 10(8) 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.
Publisher: SPRINGER VERLAG
Keywords: COLONY-STIMULATING FACTOR; LEUKEMIA INHIBITORY FACTOR; AMINO-ACID SEQUENCE; NEUTRALIZING MONOCLONAL-ANTIBODIES; IONIZATION-MASS-SPECTROMETRY; IL-6 SIGNAL TRANSDUCER; RECEPTOR-BINDING SITE; BIOLOGICAL-ACTIVITY; GROWTH-FACTOR; MOLECULAR-CLONING
Publisher's Version: https://doi.org/10.1111/j.1432-1033.1992.tb17123.x
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Creation Date: 1992-08-01 12:00:00