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

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Abstract

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 IC50 of rFIP-gap1 towards A549 and HeLa cancer cells were 29.89 and 8.34 μg/mL, respectively, whereas IC50 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.

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Acknowledgments

This work was supported mainly by a grant from National Natural Science Foundation of China (Grant No. 31000928) and also by two other grants from the Cultivation Plan for Youth Agricultural Science and Technology Innovative Talents of Liaoning Province (Grant No. 2015043) as well as Technology Pillar Program of Liaoning Province, China (Grant No. 2015103001).

Funding

This study was supported mainly by a grant from National Natural Science Foundation of China (31000928) and two other grants from the Cultivation Plan for Youth Agricultural Science and Technology Innovative Talents of Liaoning Province (2015043) as well as Technology Pillar Program of Liaoning Province (2015103001).

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  1. Siya Zhou, Shixin Guan and Zuowen Duan contributed equally to this work.

Authors and Affiliations

  1. College of Bioscience and Biotechnology, Shenyang Agricultural University, Shenyang, 110866, China

    Siya Zhou, Zuowen Duan, **ao Han, **n Zhang, Haoge Li, Li**g Chen, Hui Ma, Yanye Ruan & **gwei Lin

  2. Liaoning Province Key Laboratory of Agricultural Technology, Dongling Road, 120, Shenyang, 110866, Liaoning, China

    Siya Zhou, Zuowen Duan, **ao Han, **n Zhang, Haoge Li, Li**g Chen, Hui Ma, Yanye Ruan & **gwei Lin

  3. Liaoning Province Research Centre of Plant Genetic Engineering Technology, Dongling Road, 120, Shenyang, 110866, Liaoning, China

    Siya Zhou, Zuowen Duan, **ao Han, **n Zhang, Haoge Li, Li**g Chen, Hui Ma, Yanye Ruan & **gwei Lin

  4. College of Horticulture, Shenyang Agricultural University, Shenyang, 110866, China

    Shixin Guan & Wenli Fan

  5. China Meitan General Hospital, Bei**g, 100028, China

    Hangmei Liu

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  1. Siya Zhou
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Zhou, S., Guan, S., Duan, Z. et al. Molecular cloning, codon-optimized gene expression, and bioactivity assessment of two novel fungal immunomodulatory proteins from Ganoderma applanatum in Pichia. Appl Microbiol Biotechnol 102, 5483–5494 (2018). https://doi.org/10.1007/s00253-018-9022-5

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