Expression of biologically active murine interleukin-18 in Lactococcus lactis

• Published in: FEMS microbiology letters 2016-11, Vol.363 (21), p.fnw234
• Main Authors: Feizollahzadeh, Sadegh, Khanahmad, Hossein, Rahimmanesh, Ilnaz, Ganjalikhani-hakemi, Mazdak, Andalib, Alireza, Sanei, Mohammad Hossein, Rezaei, Abbas
• Format: Article
• Language: English
• Subjects: Animals; Biological activity; Cells, Cultured; Cytokines; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Gel electrophoresis; Gram-positive bacteria; Immune response; Immune system; Industrial applications; Interferon; Interferon-gamma - biosynthesis; Interleukin 18; Interleukin-18 - biosynthesis; Interleukin-18 - genetics; Interleukin-18 - metabolism; Lactococcus lactis; Lactococcus lactis - genetics; Lactococcus lactis - metabolism; Lymphocytes; Lymphocytes T; Lysates; Mice; Microbiology; Mucosal immunity; Mucous Membrane - immunology; Mucous Membrane - microbiology; Nisin; Nisin - genetics; Plasmids - genetics; Promoter Regions, Genetic - genetics; Protein expression; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Sodium lauryl sulfate; Spleen; Western blotting
• ISSN: 0378-1097; 1574-6968; 1574-6968
• DOI: 10.1093/femsle/fnw234

Abstract The food-grade bacterium Lactococcus lactis is increasingly used for heterologous protein expression in therapeutic and industrial applications. The ability of L. lactis to secrete biologically active cytokines may be used for the generation of therapeutic cytokines. Interleukin (IL)-18 enhances the immune response, especially on mucosal surfaces, emphasizing its therapeutic potential. However, it is produced as an inactive precursor and has to be enzymatically cleaved for maturation. We genetically manipulated L. lactis to secrete murine IL-18. The mature murine IL-18 gene was inserted downstream of a nisin promoter in pNZ8149 plasmid and the construct was used to transform L. lactis NZ3900. The transformants were selected on Elliker agar and confirmed by restriction enzyme digestion and sequencing. The expression and secretion of IL-18 protein was verified by SDS-PAGE, western blotting and ELISA. The biological activity of recombinant IL-18 was determined by its ability to induce interferon (IFN)-γ production in L. lactis co-cultured with murine splenic T cells. The amounts of IL-18 in bacterial lysates and supernatants were 3–4 μg mL−1 and 0.6–0.7 ng mL−1, respectively. The successfully generated L. lactis strain that expressed biologically active murine IL-18 can be used to evaluate the possible therapeutic effects of IL-18 on mucosal surfaces. This study reports the construction and use of genetically manipulated Lactococcus lactis for efficient production of active IL-18. Graphical Abstract Figure. This study reports the construction and use of genetically manipulated Lactococcus lactis for efficient production of active IL-18.