Production and functional characterization of a novel fungal immunomodulatory protein FIP-SN15 shuffled from two genes of Ganoderma species
- 462 Downloads
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.
KeywordsFungal immunomodulatory protein (FIP) Expression Apoptosis Cell cycle Fluorescence activated cell sorting (FACS)
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).
- Kong XH, Zhang JC, Han X, Zhang PQ, Dai XD, Liu JN, Zhang XX, Lee I, Liu SK (2013) High-yield production in Escherichia coli of fungal immunomodulatory protein isolated from Flammulina velutipes and its bioactivity assay in vivo. Int J Mol Sci 14:2230–2241. doi: 10.3390/ijms14022230 PubMedCentralPubMedCrossRefGoogle Scholar
- Li QZ, Huang L, Xie MQ, Zhou XW (2009) Cloning, expression, and purification of a fungal immunomodulatory protein from Ganoderma lucidum. The 5th International Medicinal Mushroom Conference, 338–346 (Nantong, Jiangsu, China)Google Scholar
- Li SY, Zhao ZL, Li Y, Tang XM (2012b) Advances of fungal immunomodulatory proteins in macromycete. Biotech Bulletin 5:49–53 (in Chinese)Google Scholar
- Lin WH, Hung CH, Hsu CI, Lin JY (1997) Dimerization of the N-terminal amphipathic a-Helix domain of the fungal immunomodulatory protein from Ganoderma tsugae (Fip-gts) defined by a yeast two-hybrid system and site-directed mutagenesis. J Biol Chem 272:20044–20048. doi: 10.1074/jbc.272.32.20044 PubMedCrossRefGoogle Scholar
- Lin JW, Sun F, Zhang R, Zhang CY, Liu LX (2005) Advances of fungal immunomodulatory proteins. Chin J Microbiol Immunol 21:477–480 (in Chinese)Google Scholar
- Wang XF, Su KQ, Bao TW, Cong WR, Chen YF, Li QZ, Zhou XW (2012) Immunomodulatory effects of fungal proteins. Curr Top Nutraceut R 10:1–11Google Scholar
- Wu MY, Hsu MF, Huang CS, Fu HY, Huang CT (2007) A 2.0Å structure of GMI, a member of the fungal immunomodulatory protein family from Ganoderma microsporum. Protein Crystallogr II:132Google Scholar
- Zhou XW, Wang XF, Li QZ, Su KQ, Cong WR (2011) A nucleotide sequence enconding fungal immunomodulatory protein shuffled from Ganoderma lucidum and Ganoderma sinensis, the preparation method of recombinant protein and its application. Chinese patent no. ZL 2011 1 0068813.0 (In Chinese)Google Scholar