High yield refolding and purification process for recombinant human interleukin-6 expressed in Escherichia coli

Recombinant human interleukin-g (hIL-6), a pleiotropic cytokine containing two intramolecular disulfide bonds, was expressed in Escherichia coil as an insoluble inclusion body, before being refolded and purified in high yield providing sufficient qualities for clinical use. Quantitative reconstituti on of the native disulfide bonds of hIL-6 from the fully denatured E. coli extracts could be performed by glutathione-assisted oxidation in a complete ly denaturating condition (6M guanidinium chloride) at protein concentratio ns higher than 1 mg/mL, preventing aggregation of reduced hIL-6. Oxidation in 6M guanidinium chloride (GdnHCl) required remarkably low concentrations of glutathione (reduced form, 0.01 mM; oxidized form, 0.002 mM) to be added to the solubilized hIL-6 before the incubation at pH 8.5, and 22 degrees C for 16 h. After completion of refolding by rapid transfer of oxidized hIL- 6 into acetate buffer by gel filtration chromatography, residual contaminan ts including endotoxin and E. coli proteins were efficiently removed by suc cessive steps of chromatography. The amount of dimeric hIL-6s, thought to b e purification artifacts, was decreased by optimizing the salt concentratio ns of the loading materials in the ion-exchange chromatography, and gradual ly removing organic solvents from the collected fractions of the preparativ e reverse-phase HPLC. These refolding and purification processes, which giv e an overall yield as high as 17%, seem to be appropriate for the commercia l scale production of hIL-6 for therapeutic use. (C) 1999 John Wiley & Sons , Inc.