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Int. J. Mol. Sci. 2018, 19(7), 1933; https://doi.org/10.3390/ijms19071933

p19-Targeting ILP Protein Blockers of IL-23/Th-17 Pro-Inflammatory Axis Displayed on Engineered Bacteria of Food Origin

1
Department of Biotechnology, Jožef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia
2
Graduate School of Biomedicine, Faculty of Medicine, University of Ljubljana, SI-1000 Ljubljana, Slovenia
3
Laboratory of Ligand Engineering, Institute of Biotechnology of the Czech Academy of Sciences, v. v. i., BIOCEV Research Center, Průmyslová 595, 252 50 Vestec, Czech Republic
4
Laboratory of Structural Bioinformatics of Proteins, Institute of Biotechnology of the Czech Academy of Sciences, v. v. i., BIOCEV Research Center, Průmyslová 595, 252 50 Vestec, Czech Republic
5
Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, SI-1000 Ljubljana, Slovenia
*
Author to whom correspondence should be addressed.
Received: 17 May 2018 / Revised: 23 June 2018 / Accepted: 29 June 2018 / Published: 1 July 2018
(This article belongs to the Special Issue The Interleukins in Health and Disease)
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Abstract

IL-23-mediated Th-17 cell activation and stimulation of IL-17-driven pro-inflammatory axis has been associated with autoimmunity disorders such as Inflammatory Bowel Disease (IBD) or Crohn’s Disease (CD). Recently we developed a unique class of IL-23-specific protein blockers, called ILP binding proteins that inhibit binding of IL-23 to its cognate cell-surface receptor (IL-23R) and exhibit immunosuppressive effect on human primary blood leukocytes ex vivo. In this study, we aimed to generate a recombinant Lactococcus lactis strain which could serve as in vivo producer/secretor of IL-23 protein blockers into the gut. To achieve this goal, we introduced ILP030, ILP317 and ILP323 cDNA sequences into expression plasmid vector containing USP45 secretion signal, FLAG sequence consensus and LysM-containing cA surface anchor (AcmA) ensuring cell-surface peptidoglycan anchoring. We demonstrate that all ILP variants are expressed in L. lactis cells, efficiently transported and secreted from the cell and displayed on the bacterial surface. The binding function of AcmA-immobilized ILP proteins is documented by interaction with a recombinant p19 protein, alpha subunit of human IL-23, which was assembled in the form of a fusion with Thioredoxin A. ILP317 variant exhibits the best binding to the human IL-23 cytokine, as demonstrated for particular L.lactis-ILP recombinant variants by Enzyme-Linked ImmunoSorbent Assay (ELISA). We conclude that novel recombinant ILP-secreting L. lactis strains were developed that might be useful for further in vivo studies of IL-23-mediated inflammation on animal model of experimentally-induced colitis. View Full-Text
Keywords: lactococcus; binding protein; albumin-binding domain; cytokine; IL-23; surface display lactococcus; binding protein; albumin-binding domain; cytokine; IL-23; surface display
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).
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Škrlec, K.; Zadravec, P.; Hlavničková, M.; Kuchař, M.; Vaňková, L.; Petroková, H.; Křížová, L.; Černý, J.; Berlec, A.; Malý, P. p19-Targeting ILP Protein Blockers of IL-23/Th-17 Pro-Inflammatory Axis Displayed on Engineered Bacteria of Food Origin. Int. J. Mol. Sci. 2018, 19, 1933.

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