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.

Interferon Beta Activity Is Modulated via Binding of Specific S100 Proteins

“…Homogeneity of S100 protein samples was controlled by SDS-PAGE. Their molecular masses were verified by electrospray ionization mass spectrometry as previously described [ 65 ].…”

Cloning and Expression of Interleukin 11 in Bacillus subtilis

“…The aim of this study is cloning and expression of Interleukin 11 in Bacillus subtilis in order to produce this valuable cytokine in lab scale. Till now, the gene of IL-11 has been cloned and expressed in different pro-and eukaryotic hosts such as human endometrium, tobacco and E. coli (Karpovich et al, 2003;Sadeghi et al, 2016;Sokolov et al, 2016). Here, for the first time the gene of IL-11 is cloned and expressed in B. subtilis.…”

Recognition of granulocyte-macrophage colony-stimulating factor by specific S100 proteins

“…The fractions corresponding to GM-CSF were collected, concentrated to a concentration of 1.5 mg/mL and stored at −70°C. The mass of the GM-CSF sample corresponds to residues 18-144 of UniProt ID P04141, which was confirmed by electrospray ionization mass spectrometry according to [54]. Functional activity of the GM-CSF sample was verified using an MTT assay as described below.…”