Abstract: The mucin-degrading gut commensal bacterium Akkermansia muciniphila has emerged as a promising probiotic due to its significant health-promoting effects, wherein its protein constituents mediate critical host-microbe crosstalk. Notably, Amuc_0119 represents an uncharacterized protein potentially with beneficial biological functions. In this study, we aimed to construct an expression system for recombinant Amuc_0119 to facilitate its structural and functional characterization. The coding sequence of Amuc_0119 was cloned into pET-28a(+) vector with an N-terminal 6×His-tag and successfully transformed into E. coli BL21(DE3) competent cells. Protein expression was induced by 0.5 mM IPTG overnight at 37°C. SDS-PAGE analysis revealed successful expression of the 46 kDa recombinant protein, which was subsequently purified via Ni-NTA affinity chromatography. Western blot with anti-His antibodies confirmed target protein identity, while quantitative BCA assay determined a final concentration of 818.44 μg/mL. This study establishes the first efficient expression and purification protocol for Akkermansia muciniphila-derived Amuc_0119, providing essential tools for forthcoming structural studies and functional investigations for this bacterial protein.

Key words: Akkermansia muciniphila, Probiotic, Amuc_0119, Expression, Purification