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Applied Microbiology and Biotechnology

, Volume 102, Issue 13, pp 5483–5494 | Cite as

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

  • Siya Zhou
  • Shixin Guan
  • Zuowen Duan
  • Xiao Han
  • Xin Zhang
  • Wenli Fan
  • Haoge Li
  • Lijing Chen
  • Hui Ma
  • Hangmei Liu
  • Yanye Ruan
  • Jingwei Lin
Biotechnological products and process engineering
  • 53 Downloads

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.

Keywords

Ganoderma applanatum Fungal immunomodulatory protein Codon optimization Yeast expression Bioactivities 

Notes

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).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2018_9022_MOESM1_ESM.pdf (1.2 mb)
ESM 1 (PDF 1242 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Siya Zhou
    • 1
    • 2
    • 3
  • Shixin Guan
    • 4
  • Zuowen Duan
    • 1
    • 2
    • 3
  • Xiao Han
    • 1
    • 2
    • 3
  • Xin Zhang
    • 1
    • 2
    • 3
  • Wenli Fan
    • 4
  • Haoge Li
    • 1
    • 2
    • 3
  • Lijing Chen
    • 1
    • 2
    • 3
  • Hui Ma
    • 1
    • 2
    • 3
  • Hangmei Liu
    • 5
  • Yanye Ruan
    • 1
    • 2
    • 3
  • Jingwei Lin
    • 1
    • 2
    • 3
  1. 1.College of Bioscience and BiotechnologyShenyang Agricultural UniversityShenyangChina
  2. 2.Liaoning Province Key Laboratory of Agricultural TechnologyShenyangChina
  3. 3.Liaoning Province Research Centre of Plant Genetic Engineering TechnologyShenyangChina
  4. 4.College of HorticultureShenyang Agricultural UniversityShenyangChina
  5. 5.China Meitan General HospitalBeijingChina

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