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Purification and characterization of a recombinant murine interleukin-6.
- Source :
- European Journal of Biochemistry; 8/1/92, Vol. 207 Issue 3, p903-913, 11p
- Publication Year :
- 1992
-
Abstract
- Murine interleukin-6 (IL-6), when expressed in Escherichia coil 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 sohibilized 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°C. About 25 mg pure protein was obtained from 37 g wet cells. This recombinant murine 1L-6 had a specific activity in the hybridoma growth factor assay of 2×10<superscript>8</superscript> 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 inachve in the hybridoma growth factor assay. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 00142956
- Volume :
- 207
- Issue :
- 3
- Database :
- Complementary Index
- Journal :
- European Journal of Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 13670631
- Full Text :
- https://doi.org/10.1111/j.1432-1033.1992.tb17123.x