Application of combined reagent solution to the oxidative refolding of recombinant human interleukin 6

Human interleukin 6 (hIL-6), which is a cytokine involved in diverse biolog ical activities, consists of a four-helix bundle with two disulfide bonds. For the clinical use of hIL-6 in cancer therapy, designing of commercial-sc ale production systems of recombinant hIL-6 (rhIL-6) expressed by E. coli h as been attempted. Since rhIL-6 has been produced as inclusion bodies in th e expression systems reported to date, establishment of a strategy to achie ve a high yield of refolding of this recombinant protein is quite desirable . It has been reported that oxidation of rhIL-6 under a completely denaturi ng condition suppresses aggregation during the refolding process [Ejima et al., Biotechnol. Bioeng., 62. 301-310 (1999)]. In this protocol, however, s mall but significant amounts of unidentified by-products unavoidably arose, which might be problematic in the therapeutic use of rhIL-6. In the presen t study, detailed characterization of the individual by-products has been p erformed on inspection of peptide maps, and the by-products found to origin ate from improperly formed disulfide bonds, most of which are disulfide-lin ked dimers. In order to minimize these by-products, combined solutions of u rea and LiCl were used for oxidative refolding of rhIL-6. It was demonstrat ed that combined use of 1-2 m urea and 1-3 m LiCl effectively suppresses th e formation of the by-products as well as aggregates. We propose that the u se of the combined reagents can be an alternative method for refolding of r hIL-6 for clinical purposes.