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.

Department of Biotechnology Jožef Stefan Institute Jamova 39 SI 1000 Ljubljana Slovenia

Department of Biotechnology Jožef Stefan Institute Jamova 39 SI 1000 Ljubljana Slovenia Faculty of Pharmacy University of Ljubljana Aškerčeva 7 SI 1000 Ljubljana Slovenia

Department of Biotechnology Jožef Stefan Institute Jamova 39 SI 1000 Ljubljana Slovenia Graduate School of Biomedicine Faculty of Medicine University of Ljubljana SI 1000 Ljubljana Slovenia

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

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

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