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The fungal immunomodulatory proteins (FIPs) are a new protein family identified from several edible and medical mushrooms and play an important role in antitumor, anti-allergy and immunomodulating activities. A gene encoding the FIP was cloned from the mycelia of Changbai Lingzhi (Ganoderma lucidum) and recombinant expressed in the Pichia pastoris expression system. SDS-PAGE, amino acid composition and circular dichroism analyses of the recombinant FIP (reFIP) indicated that the gene was correctly and successfully expressed. In vitro assays of biological activities revealed that the reFIP exhibited similar immunomodulating capacities as native FIPs. The reFIP significantly stimulated the proliferation of mouse spleen lymphocytes and apparently enhanced the expression level of interleukin-2 released from the mouse splenocytes. In addition, anti-tumor activity assay showed that the reFIP could inhibit the proliferation of human leukemia-NB4 by inducing the cell apoptosis to a degree of about 32.4%. Taken together, the FIP gene from Changbai G. lucidum has been integrated into the yeast genome and expressed effectively at a high level (about 191.2 mg l -1 ). The reFIP possessed very similar biological activities to native FIPs, suggesting its potential application as a food supplement or immunomodulating agent in pharmaceuticals and even medical studies.

BACKGROUND Fungal immunomodulatory proteins (FIPs) are a newly identified protein family that exhibits immunomodulation and antitumor activities. However, little is known about the FIPs from Gomphidiaceae species. In this study, a novel FIP gene termed FIP‐cru was cloned from Chroogomphis rutilus, analyzed, and functionally expressed in Pichia pastoris GS115. In addtion, the bioactivity of rFIP‐cru was examined including hemagglutination, cytotoxicity, and interleukin‐2 assays in vitro. RESULTS FIP‐cru comprised 342 bp and encoded a peptide of 113 amino acids, which shared a high homology to other FIPs. rFIP‐cru was obtained by expressing the recombinant gene in P. pastoris GS115, with a yield of 148.5 mg L−1, and then purified via ammonium sulfate precipitation and agarose nickel magnetic nanobeads. The bioactivity assay showed that rFIP‐cru can agglutinate mouse and sheep red blood cells but not human cells. Additionally, rFIP‐cru can stimulate the proliferation of murine splenocytes and enhance the secretion of interleukin‐2. CONCLUSION FIP‐cru is a novel fungal immunomodulatory protein. The use of P. pastoris as an expression system will provide a feasible production method to obtain large amounts of rFIP‐cru to study its potential application in therapeutic and medical studies. © 2016 Society of Chemical Industry

Fungal immunomodulatory proteins (FIPs) have been identified from a series of fungi, especially in Ganoderma species. However, little is known about the FIPs from G. applanatum. In this study, two novel FIP genes, termed as FIP-gap1 and FIP-gap2, were cloned from G. applanatum, characterized and functionally expressed after codon optimization in Pichia pastoris GS115. Results showed that FIP-gap1 and FIP-gap2 comprised 342-bp encoding peptides of 113 amino acids, which shared a high homology with other Ganoderma FIPs. The yield of recombinant FIP-gap1 and FIP-gap2 increased significantly after codon optimization and reached 247.4 and 197.5 mg/L, respectively. Bioactivity assay in vitro revealed that both rFIP-gap1 and rFIP-gap2 could agglutinate mouse, sheep, and human red blood cells. Besides, rFIP-gap1 and rFIP-gap2 obviously stimulated the proliferation of mouse splenocytes and enhanced IL-2 and IFN-γ release. Cytotoxicity detection indicated that IC of rFIP-gap1 towards A549 and HeLa cancer cells were 29.89 and 8.34 μg/mL, respectively, whereas IC of rFIP-gap2 to the same cancer cells were 60.92 and 41.05 μg/mL, respectively. Taken together, novel FIP gaps were cloned and functionally expressed in P. pastoris, which can serve as feasible and stable resources of rFIP gaps for further studies and potential applications.