Recombinant production of human interleukin 6 in Escherichia coli.
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).
Department of Agrobiotechnology, Agricultural and Environmental Faculty, University of Rostock, Rostock, Germany.
Henrik
H
Nausch
Jana
J
Huckauf
Roswitha
R
Koslowski
Udo
U
Meyer
Inge
I
Broer
Heike
H
Mikschofsky
D000595:Amino Acid Sequence; D001483:Base Sequence; D003593:Cytoplasm / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects; D023061:Gene Order; D006801:Humans; D015850:Interleukin-6 / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D018832:Molecular Chaperones / Q000096:biosynthesis; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins; D013696:Temperature
D015850:Interleukin-6; D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins; D007544:Isopropyl Thiogalactoside