Lunasin is a novel promising health-beneficial peptide derived from soybean. However, the application of lunasin is limited by its high cost. In this study, we developed a successful protocol for expression of a dimer formation protein containing 4 tandem repeated lunasin analogs (lunasin-4) in Pichia pastoris. The expression level at the optimal condition (initial pH 7.0,1.0% final methanol concentration and induction for 72 h at 26 degrees C) was 0.24 mg/mL cell-free broth. Lunasin analog, obtained from purified lunasin-4 protein through enterokinase digestion and ultrafiltration, significantly decreased (p < 0.05) the release of nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages in a dose-dependent manner. In addition, intracellular signaling array analysis demonstrated down-regulated levels of phosphorylated Akt, mechanistic target of rapamycin (mTOR) and p70 s6 kinase (p70s6k) and an up-regulated level of glycogen synthase kinase-3 beta (GSK-3 beta) after lunasin analog treatment. These results suggest that lunasin analog exerted anti-inflammatory activities in LPS-stimulated RAW264.7 cells partly via inhibiting the activation of Ala/mTOR/p70s6k signaling pathway. In conclusion, this study provides a potential strategy for recombinant production of bioactive lunasin in industry. (C) 2018, The Society for Biotechnology, Japan. All rights reserved.