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Applied Microbiology and Biotechnology

, Volume 98, Issue 13, pp 5967–5975 | Cite as

Production and functional characterization of a novel fungal immunomodulatory protein FIP-SN15 shuffled from two genes of Ganoderma species

  • Wei-ran Cong
  • Hui Xu
  • Yan Liu
  • Qi-zhang Li
  • Wei Li
  • Xuan-wei Zhou
Biotechnologically relevant enzymes and proteins

Abstract

Fungal immunomodulatory protein (FIP), extracted from higher basidiomycetes, is a kind of small molecule protein with extensive biological functions, including anti-tumor and anti-allergy, stimulating immune cells to produce a variety of cytokines, etc. Compared with FIP-glu, FIP-SN15, a novel gene shuffled from the genes of Ganoderma sinensis and Ganoderma lucidum FIP, was used as the object in this study. Based on the construction of prokaryotic expression vectors, both pET30a-FIP-glu and pET30a-FIP-SN15 were expressed in Escherichia coli. Then the recombinant proteins are respectively analyzed by Western blot, Q-TOF MS, and bioinformatics techniques. Finally, effects of reFIPs on cell cycle and apoptosis of human glioblastoma cell line U-251 MG were studied by fluorescence activated cell sorting (FACS). The results showed that the recombinant proteins FIP-SN15 and FIP-glu could be successfully expressed in E. coli, the yield of which was 35.95 and 36.67 mg/L, respectively. The recombinant protein FIP-SN15 consisted of 111 amino acids, and four peptides were identified by Q-TOF MS with a coverage of 91.9 %. The secondary and tertiary structure of FIP-SN15 were also predicted by bioinformatics method which suggest that reFIP-SN15 was a new member of FIPs family. FACS analysis showed that 10 μg/mL FIP-SN15 and FIP-glu could induce U-251 MG cells apoptosis, the apoptotic rates were increased by 6.03 and 22.01 %, respectively. The results of reFIPs on U-251 MG cell cycle indicated that reFIPs could inhibit cell cycle progression by retardation of G1/S transition. The efforts in this assay would lay the foundation for further development of reFIPs products and research on the anti-tumor mechanisms of FIP-SN15.

Keywords

Fungal immunomodulatory protein (FIP) Expression Apoptosis Cell cycle Fluorescence activated cell sorting (FACS) 

Notes

Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (no: 30771500), Shanghai Science and Technology Committee and Shanghai Leading Academic Discipline Project and Shanghai Natural Science Foundation (no. 11ZR1416800).

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Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  1. 1.Plant Biotechnology Research Center, Shanghai Key Laboratory of Agro-biotechnology, Key Laboratory of Urban Agriculture (South) Ministry of Agriculture, School of Agriculture and BiologyShanghai Jiao Tong UniversityShanghaiPeople’s Republic of China
  2. 2.Key Laboratory of Systems Biomedicine, Ministry of Education, Shanghai Center for Systems BiomedicineShanghai Jiao Tong UniversityShanghaiPeople’s Republic of China

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