Characterization and anticancer bioactivity of the fungal immunomodulatory protein FIP-Gre from Ganoderma resinaceum

Background: Natural products remain rich sources of anticancer agents. Fungal immunomodulatory proteins (FIPs) from medicinal mushrooms show direct in vitro tumor inhibition; we investigated a new FIP, FIP-Gre, from Ganoderma resinaceum . Methods: FIP-Gre was identified by genome mining (LZ8 query), modeled by AlphaFold2, and assessed by docking/100-ns MD (EGFR, c-Met). Recombinant FIP-Gre was produced in E. coli BL21 (DE3)/pET-29a (+), purified by Ni-NTA (~12.5 kDa), and tested for hemagglutination, antibacterial activity, cytotoxicity (A549, HepG2, MCF-7; HEK-293 control), and apoptosis markers in A549 cells (8 µg/mL). Results: FIP-Gre shares 90% identity with LZ8, contains a conserved Fve-type domain, and adopts a stable β-strand–rich fold. Docking/MD suggest strong, stable binding to EGFR and c-Met. FIP-Gre agglutinated mouse RBCs at 4 µg/mL, showed no antibacterial activity, and selectively inhibited cancer cells with IC₅₀ values of 21.27µg/mL (A549), 29.96µg/mL (HepG2), and 43.34µg/mL (MCF-7) while showing no toxicity toward normal HEK-293 cells. At 8 µg/mL, FIP-Gre significantly induced apoptosis in A549 cells by upregulating Bax, Caspase-3, Caspase-9, and p53, and downregulating Bcl-2 , indicating activation of the mitochondrial apoptotic pathway. Conclusion: FIP-Gre is a structurally robust, mushroom-derived protein with selective in vitro anticancer activity and predicted RTK engagement, supporting its development, particularly against lung carcinoma.