Expression, Purification, and Characterization of Interleukin-11 Orthologues

Abstract

Interleukin-11 (IL-11) is a multifunctional cytokine implicated in several normal and pathological processes. The decoding of IL-11 function and development of IL-11-targeted drugs dictate the use of laboratory animals and need of the better understanding of species specificity of IL-11 signaling. Here, we present a method for the recombinant interleukin-11 (rIL-11) production from the important model animals, mouse and macaque. The purified mouse and macaque rIL-11 interact with extracellular domain of human IL-11 receptor subunit α and activate STAT3 signaling in HEK293 cells co-expressing human IL-11 receptors with efficacies resembling those of human rIL-11. Hence, the evolutionary divergence does not impair IL-11 signaling. Furthermore, compared to human rIL-11 its macaque orthologue is 8-fold more effective STAT3 activator, which favors its use for treatment of thrombocytopenia as a potent substitute for human rIL-11. Compared to IL-6, IL-11 signaling exhibits lower species specificity, likely due to less conserved intrinsic disorder propensity within IL-6 orthologues. The developed express method for preparation of functionally active macaque/mouse rIL-11 samples is suited for exploration of the molecular mechanisms underlying IL-11 action and for development of the drug candidates for therapy of oncologic/hematologic/inflammatory diseases related to IL-11 signaling.

Keywords: STAT3 signaling; bacterial expression; cloning; cytokines; interleukin-11; ligand-receptor interaction; protein-protein interaction; ubiquitin.

Figure 1

Crystal structure of human IL-11 (PDB entry 4MHL [20]). α-Helices A to D are labelled. Dashed lines mark the non-resolved regions. The residues comprising the proposed sites for IL-11 interaction with IL-11Rα (site I, green) and gp130 (site II, yellow; site III, grey) receptors [20] are indicated. The residues not conserved in either macaque or mouse IL-11 are shown in cyan (see Figure S1).

Figure 2

(A) Plasmid map of pHUE IL-11 vector showing the Ub coding region (black arrow), the IL-11 coding region (grey arrow), the T7 RNA polymerase promoter (black triangle) and other regions (shaded arrows), including ColE1 origin of replication (Ori), LacI repressor (LacI) and β-lactamase (Amp) genes. Arrows indicate the direction of transcription. The SacII/NotI restriction sites used for cloning of IL-11 gene are shown; (B) The schematic presentation of the Ub-IL-11 fusion protein. The black arrow indicates the site of hydrolysis by Usp2 protease. The nucleotide sequences of the 5′ end and the restrictions sites (macaque IL-11) are shown. The translation into the protein sequence is shown below. The numbering of protein residues corresponds to the non-processed protein (Swiss-Prot entry P20808).

Figure 3

(A) Expression in E. coli BL21(DE3)/pLacIRARE strain and purification of murine IL-11 (the data for macaque IL-11 are similar), controlled by reducing SDS-PAGE (15%; staining by Coomassie brilliant blue R-250). Lane 1, soluble extract of the cells expressing the Ub-IL-11 fusion; lanes 2 and 3, the affinity-purified Ub-IL-11 fusion sample before and after the cleavage by Usp2 protease, respectively; lane 4, the IL-11 sample after cleaning from ubiquitin and Usp2 protease; lanes 5–10, the protein fractions after the gel filtration using Sephacryl S-100 HR medium (the sample shown in lane 5 was discarded). ‘M’ denotes the lanes with molecular mass standards (the masses in kDa are indicated in-between the panels A and B); (B) SDS-PAGE of the purified recombinant human, macaque and murine IL-11 (lanes 1, 2 and 3, respectively).

Figure 4

ESI-MS spectra of macaque (A) and murine (B) rIL-11 samples. m/z values and their respective relative charge values (+z) are indicated above the major peaks.

Figure 5

Kinetics of the interaction of macaque/murine rIL-11 with sIL-11Rα (panels A and B, respectively) at 25 °C, monitored by SPR spectroscopy (PBS, 0.05% TWEEN 20, pH 7.4 buffer). The biotinylated rIL-11 was immobilized on the surface of NLC sensor chip. Concentrations of sIL-11Rα used as an analyte are indicated above the curves. Grey curves are experimental, while black curves are theoretical, calculated according to the heterogeneous ligand model (1) (see Table 1 for the fitting parameters).

Figure 6

Predictions of per-residue disorder level for mature IL-11 (panel A) and IL-6 (panel B) orthologues from human (black solid lines), macaque (red dashed lines), and mouse (green dashed lines), using PONDR^® VLXT algorithm (http://pondr.com/). Positions of sites responsible for recognition of IL-receptors are shown as differently colored vertical bars (pink, cyan, and blue for sites I, II, and III, respectively).

Figure 7

IL-11-induced STAT3 activation in a HEK-Blue™ IL-6 cell line transfected with human IL-11Rα gene, monitored by STAT3-induced secretion of SEAP (QUANTI-Blue™ assay) for murine, macaque and human rIL-11. Standard deviations estimated from the duplicate measurements are shown. The dashed curves are theoretical fits to the experimental curves using Boltzmann function.