Rat interleukin 6: Expression in recombinant Escherichia coli, purification and development of a novel ELISA

Interleukin 6 (IL-6) is a cytokine involved in many aspects of the acute ph ase and immune reponses. Cloning of rat IL-6 cDNA into the pET-21d expressi on plasmid under control of a bacteriophage T7 RNA polymerase promoter syst em allowed isopropylthio-galactopyranoside (IPTG)-inducible production of r ecombinant rat IL-6 in Escherichia coli. The cloning, expression and purifi cation of rat IL-6 is described. In this expression system, rat IL-6 was pr oduced in insoluble inclusion bodies. The protein was solubilized in 6 M gu anidine hydrochloride and refolded in a glutathione redox system. Refolded rat IL-6 was purified to homogeneity using anion-exchange chromatography on SP-Trisacryl. The purified recombinant rat IL-6 had a molecular mass of 21 756.38 +/- 0.25 Da, which is within 0.01% of the predicted value, taking i nto account cleavage of the N-terminal methionine residue and the formation of two disulfide bridges. Recombinant rat IL-6 was 2-3-fold more bioactive than the human standard preparation in the B9 hybridoma bioassay. Purified rat IL-6 was used to raise polyclonal antibodies in sheep and these reagen ts were used to develop a novel rat IL-6 enzyme-linked immunosorbent assay (ELISA). The ELISA is sensitive to 10 pg/ml and has been shown to detect IL -6 in plasma from rats injected with lipopolysaccaride (LPS). (C) 1999 Acad emic Press.