Figure 1.
Constructs used to express the IL6 gene.
(A) Schematic representations of the of the vector; The vector backbones encode a kanamycin resistance gene and the pBR322 origin of replication. GST™-Tag: glutathione S-transferase (220 aa) from Schistosoma japanicum; His-Tag: 6 Histidine residues; T7-Tag®: initial 11 aa of the gene 10 protein from the T7-phage; phoA: periplasmic targeting signal of the E. coli alkaline phosphatase. Thrombin: 6 aa comprising a cleavage site for the thrombin protease. IL6ΔSig (184 aa) synthetic, codon-optimized gene sequences based on the mature protein sequence (without the ER-targeting signal); (B) Synthetic, codon-optimized coding region of the human cytokine IL6.
Table 1.
Recombinant strains used in this work.
Figure 2.
Western blotting analysis of the IL6-expressing E. coli strains BL21 and Origami 2.
(S) soluble and (I) insoluble fraction of IL6.
Figure 3.
Western blotting analysis of the IL6 expression in the cytoplasm and periplasm of E. coli BL21.
(S) soluble and (I) insoluble fraction of IL6, expressed with and without the concomitant overexpression of endogenous cytoplasmic chaperones DnaK, DnaJ, GrpE, GroES, GroEL (C-Chaperones) or periplasmic chaperones DsbA, DsbC, SurA, FkpA (P-Chaperones) at 37°C and 22°C, respectively. Additionally, IL6 was expressed fused to GST in the cytoplasm.
Figure 4.
Western blotting analysis of the IL6 expression with reduced IPTG concentrations.
(S) soluble and (I) insoluble fraction of IL6, expressed with either 1 mM or 0.1 mM IPTG at 22°C in the cytoplasm of the E. coli BL21 strain with and without the concomitant overexpression of endogenous cytoplasmic chaperones or in fusion to GST.
Table 2.
Quantification of soluble IL6 by ELISA and evaluation of biological activity.
Table 3.
Evaluation of expression parameters on the solubilization of recombinant IL6 yield (-: no soluble target protein; +: <10%; ++: 10–25%; +++25–50%; ++++: >50% of total recombinant protein localized to the soluble fraction) and biological activity (n.i: not experimentally investigated.