Molecular cloning, codon-optimized gene expression, and bioactivity assessment of two novel fungal immunomodulatory proteins from Ganoderma applanatum in Pichia

• Published in: Applied microbiology and biotechnology 2018-07, Vol.102 (13), p.5483-5494
• Main Authors: Zhou, Siya, Guan, Shixin, Duan, Zuowen, Han, Xiao, Zhang, Xin, Fan, Wenli, Li, Haoge, Chen, Lijing, Ma, Hui, Liu, Hangmei, Ruan, Yanye, Lin, Jingwei
• Format: Article
• Language: English
• Subjects: Amino acids; Ascomycota; Basidiomycota; Biocompatibility; Biological activity; Biomedical and Life Sciences; Biotechnological Products and Process Engineering; Biotechnology; Cancer; Cell proliferation; Cloning; Codons; Cytotoxicity; Erythrocytes; Feasibility studies; Fungi; Ganoderma; Gene expression; Genetic aspects; Homology; Immunomodulation; Immunomodulators; Interleukin 2; Life Sciences; Microbial Genetics and Genomics; Microbiological research; Microbiology; Optimization; Peptides; Physiological aspects; Pichia pastoris; Proteins; Sheep; Splenocytes; Toxicity; Yeast; γ-Interferon
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-018-9022-5

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 50 of rFIP-gap1 towards A549 and HeLa cancer cells were 29.89 and 8.34 μg/mL, respectively, whereas IC 50 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.