Abstract
In this study, we compared basic expression approaches for the efficient expression of bioactive recombinant human interleukin-6 (IL6), as an example for a difficult-to-express protein. We tested these approaches in a laboratory scale in order to pioneer the commercial production of this protein in Escherichia coli (E. coli). Among the various strategies, which were tested under Research and Development (R&D) conditions, aggregation-prone IL6 was solubilized most effectively by co-expressing cytoplasmic chaperones. Expression of a Glutathion-S-Transferase (GST) fusion protein was not efficient to increase IL6 solubility. Alteration of the cultivation temperature significantly increased the solubility in both cases, whereas reduced concentrations of IPTG to induce expression of the T7lac-promotor only had a positive effect on chaperone-assisted expression. The biological activity was comparable to that of commercial IL6. Targeting the expressed protein to an oxidizing environment was not effective in the generation of soluble IL6. Taken together, the presence of chaperones and a lowered cultivation temperature seem effective to isolate large quantities of soluble IL6. This approach led to in vivo soluble, functional protein fractions and reduces purification and refolding requirements caused by downstream purification procedures. The final yield of soluble recombinant protein averaged approximately 2.6 mg IL6/liter of cell culture. These findings might be beneficial for the development of the large-scale production of IL6 under the conditions of current good manufacturing practice (cGMP).
Highlights
Human interleukin-6 (IL6) is a cytokine with pleiotropic functions that is involved in a broad range of biological activities and was found to be associated with a vast number of severe inflammatory diseases, sepsis and rheumatoid arthritis
The pET28-IL6DSig plasmid was introduced into the E. coli strains BL21 and Origami 2, which are characterized by either a reducing (BL21) or an oxidizing cytoplasm (Origami 2)
When expression was induced with 1 mM IPTG at 37uC for 4 h, most of the recombinant proteins were deposited in the insoluble fraction of the cell lysate both as mono- and multimeres (Fig. 2)
Summary
Human interleukin-6 (IL6) is a cytokine with pleiotropic functions that is involved in a broad range of biological activities and was found to be associated with a vast number of severe inflammatory diseases, sepsis and rheumatoid arthritis. Pilot studies demonstrated that these diseases can be efficiently treated by targeting IL6, whereas standard antibiotic-based strategies fail [1,2,3]. Functionally active recombinant expression of IL6 in large quantities is necessary, because extraction from human tissues is difficult and results in low protein yields [5]. Despite the availability and improvement of several alternative biopharmaceutical protein production platforms, E. coli offers advantages including growth on inexpensive carbon sources, rapid biomass accumulation, high cell-density fermentation and the ability to increase the production scale. E. coli is well-characterized in terms of genetics and molecular biology [6]
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