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 biological activities, consists of a four-helix bundle with two disulfide bonds. For the clinical use of hIL-6 in cancer therapy, designing of commercial-scale production systems of recombinant hIL-6 (rhIL-6) expressed by E. coli has been attempted. Since rhIL-6 has been produced as inclusion bodies in the expression systems reported to date, establishment of a strategy to achieve a high yield of refolding of this recombinant protein is quite desirable. It has been reported that oxidation of rhIL-6 under a completely denaturing condition suppresses aggregation during the refolding process [Ejima et al., Biotechnol. Bioeng., 62, 301-310 (1999)]. In this protocol, however, small but significant amounts of unidentified by-products unavoidably arose, which might be problematic in the therapeutic use of rhIL-6. In the present study, detailed characterization of the individual by-products has been performed on inspection of peptide maps, and the by-products found to originate from improperly formed disulfide bonds, most of which are disulfide-linked dimers. In order to minimize these by-products, combined solutions of urea and LiCl were used for oxidative refolding of rhIL-6. It was demonstrated that combined use of 1-2 M urea and 1-3 M LiCl effectively suppresses the formation of the by-products as well as aggregates. We propose that the use of the combined reagents can be an alternative method for refolding of rhIL-6 for clinical purposes.
Harada
T
T
Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan.
Kurimoto
E
E
Moriyama
Y
Y
Ejima
D
D
Sakai
T
T
Nohara
D
D
Kato
K
K
D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D019791:Guanidine / Q000737:chemistry; D006801:Humans; D007202:Indicators and Reagents; D015850:Interleukin-6 / Q000737:chemistry*; D018021:Lithium Chloride / Q000737:chemistry; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D012697:Serine Endopeptidases / Q000737:chemistry; D012996:Solutions; D014508:Urea / Q000737:chemistry
D004220:Disulfides; D007202:Indicators and Reagents; D015850:Interleukin-6; D011994:Recombinant Proteins; D012996:Solutions; D014508:Urea; D012697:Serine Endopeptidases; C036813:lysyl endopeptidase; D018021:Lithium Chloride; D019791:Guanidine