Rat interleukin-10: production and characterisation of biologically activeprotein in a recombinant bacterial expression system

Interleukin-10 is an anti-inflammatory Th, immunosuppressive cytokine, the active form of which is a non-covalent homodimer, and which exhibits specie s-specificity both with respect to structure and biological activity. The r at homologue of IL-10 shares 73% identity with human IL-10 at the amino-aci d sequence level, and has, in addition to the two disulphide bonds present in human IL-10, a fifth, unpaired cysteine (cys-149), Preparation of rat IL -10 by bacterial expression followed by solubilisation and refolding in a g lutathione redox system, results in a molecule in which cys-149 is almost e ntirely oxidised, existing either as disulphide dimer or as a mixed disulph ide with glutathione, and which has less than 1% of the activity of the nat ive (cys-149-SH) form of the molecule, Site directed mutagenesis of rat IL- 10 to replace cys-149 with tyrosine produces a molecule which readily adopt s the active conformation upon solubilisation and refolding, and which is r ecoverable in good yield from bacterial expression systems. Comparison of t he biological activities of rat IL-10(tyr149) and commercial rat IL-10 prep arations confirms that the activity of native-sequence rat IL-10 is either reduced or absent. It is proposed therefore that the biosynthetic analogue rat IL-10(tyr149) is a more useful molecule to investigate the biological a ctions of IL-10 in the rat.