World Journal of Microbiology and Biotechnology

, Volume 25, Issue 3, pp 383–390

Molecular cloning and recombinant expression of a gene encoding a fungal immunomodulatory protein from Ganoderma lucidum in Pichia pastoris

  • Jing-Wei Lin
  • Li-Xia Hao
  • Gui-Xue Xu
  • Fei Sun
  • Feng Gao
  • Ren Zhang
  • Li-Xia Liu
Original Paper

Abstract

The fungal immunomodulatory proteins (FIPs) are a new protein family identified from several edible and medical mushrooms and play an important role in anti-tumor, anti-allergy and immunomodulating activities. A gene encoding the FIP was cloned from the mycelia of Changbai Lingzhi (Ganoderma lucidum) and recombinant expressed in the Pichia pastoris expression system. SDS-PAGE, amino acid composition and circular dichroism analyses of the recombinant FIP (reFIP) indicated that the gene was correctly and successfully expressed. In vitro assays of biological activities revealed that the reFIP exhibited similar immunomodulating capacities as native FIPs. The reFIP significantly stimulated the proliferation of mouse spleen lymphocytes and apparently enhanced the expression level of interleukin-2 released from the mouse splenocytes. In addition, anti-tumor activity assay showed that the reFIP could inhibit the proliferation of human leukemia-NB4 by inducing the cell apoptosis to a degree of about 32.4%. Taken together, the FIP gene from Changbai G.lucidum has been integrated into the yeast genome and expressed effectively at a high level (about 191.2 mg l−1). The reFIP possessed very similar biological activities to native FIPs, suggesting its potential application as a food supplement or immunomodulating agent in pharmaceuticals and even medical studies.

Keywords

Fungal immunomodulatory protein Ganoderma lucidum Molecular cloning Recombinant expression Pichia pastoris 

Supplementary material

11274_2008_9902_MOESM1_ESM.pdf (33 kb)
MOESM1 (PDF 32 kb)

References

  1. Chang HH, Sheu F (2006) Anti-tumor mechanisms of orally administered a fungal immunomodulatory protein from Flammulina velutipes in mice. Nutr Immunol 20:A1057Google Scholar
  2. Chang S, Buswell JA (1999) Ganoderma lucidum—a mushrooming medical mushroom. Int J Med Mushrooms 1:139–146Google Scholar
  3. Chen YH, Yang JT, Chau KH (1974) Determination of the helix and β turn of proteins in aqueous solution by circular dichroism. Biochemistry 13:3350–3359. doi:10.1021/bi00713a027 CrossRefGoogle Scholar
  4. Cheng D, Huang XY, Li BJ (2002) A method for preparation of high quality DNA from medical fungi and construction of a genomic library. Mycosystema 21:137–139Google Scholar
  5. Clare JJ, Rayment FB, Ballantine SP, Sreekrishna K, Romanos MA (1991) High-level expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integrations of the gene. Biotechnology 9(5):455–460. doi:10.1038/nbt0591-455 CrossRefGoogle Scholar
  6. Haak-Frendscho M, Kino K, Sone K, Jardieu P (1993) Ling Zhi-8: a novel T cell mitogen induces cytokine production and upregulation of ICAM-1 expression. Cell Immunol 150:101–113. doi:10.1006/cimm.1993.1182 CrossRefGoogle Scholar
  7. Hsieh KY, Hsu CI, Lin JY, Tsai CC, Lin RH (2003) Oral administration of an edible-mushroom-derived protein inhibits the development of food-allergic reactions in mice. Clin Exp Allergy 33:1595–1602. doi:10.1046/j.1365-2222.2003.01790.x CrossRefGoogle Scholar
  8. Hsieh CW, Lan JL, Meng Q, Cheng YW, Huang HM, Tsai JJ (2007) Eosinophil apoptosis induced by fungal immunomodulatory peptide-fve via reducing IL-5 alpha receptor. J Formos Med Assoc 106(1):36–43CrossRefGoogle Scholar
  9. Hsu HC, Hsu CI, Lin RH, Kao CL, Lin JY (1997) Fip-vvo, a new fungal immunomodulatory protein isolated from Vovariella volvacea. Biochem J 323:557–565Google Scholar
  10. Hsu HY, Hua KF, Wu WC, Hsu J, Weng ST, Lin TL, Liu CY, Hseu RS, Huang CT (2008) Reishi immuno-modulation protein induces interleukin-2 expression via protein kinase-dependent signaling pathways within human T cells. J Cell Physiol 215:15–26. doi:10.1002/jcp.21144 CrossRefGoogle Scholar
  11. Jinn TR, Wu CM, Tu WC, Ko JL, Jason TCT (2006) Functional expression of FIP-gts, a fungal immunomodulatory protein from Ganoderma Tsugae in Sf21 insect cells. Biosci Biotechnol Biochem 70:2627–2634. doi:10.1271/bbb.60232 CrossRefGoogle Scholar
  12. Kino K, Yamashita A, Yamaoka K, Watanabe J, Tanaka S, Ko K, Shimizu K, Tsunoo H (1989) Isolation and characterization of a new immunomodulatory protein, Ling Zhi-8 (LZ-8), from Ganoderma lucidum. J Biol Chem 264:472–478Google Scholar
  13. Kino K, Mizumoto K, Sone T, Yamaji T, Watanabe J, Yamashita A, Yamaoka K, Shimizu K, Ko K, Tsunoo H (1990) An immunomodulatory protein, Ling Zhi-8 (LZ-8) prevents insulitis in nonobese diabetic mice. Diabetologia 33:713–718. doi:10.1007/BF00400340 CrossRefGoogle Scholar
  14. Ko JL, Hsu CI, Lin RH, Jai CL, Lin JY (1995) A new fungal immunomodulatory protein, FIP-fve isolated from the edible mushroom, Flammulina velutipes and its complete amino acid sequence. Eur J Biochem 228:244–249. doi:10.1111/j.1432-1033.1995.tb20256.x CrossRefGoogle Scholar
  15. Ko JL, Lin SJ, Hsu CI, Kao CL, Lin JY (1997) Molecular cloning and expression of a fungal immunomodulatory protein, FIP-fve, from Flammulina velutipes. J Formos Med Assoc 96:517–524Google Scholar
  16. Liao CH, Hsiao YM, Hsu CP, Lin MY, Wang JC, Huang YL, Ko JL (2006) Transcriptionally mediated inhibition of telomerase of fungal immunomodulatory protein from Ganoderma tsugae in A549 human lung adenocarcinoma cell line. Mol Carcinog 45(4):220–229CrossRefGoogle Scholar
  17. Liao CH, Hsiao YM, Sheu GT, Chang JT, Wang PH, Wu MF, Shieh GJ, Ko JL (2007) Nuclear translocation of telomerase reverse transcriptase and calcium signaling in repression of telomerase activity in human lung cancer cells by fungal immunomodulatory protein from Ganoderma tsugae. Biochem Pharmacol 74:1541–1554. doi:10.1016/j.bcp.2007.07.025 CrossRefGoogle Scholar
  18. Lin WH, Huang CH, Hsu CI, Lin JY (1997) Dimerization of the N-terminal amphipathic α-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 CrossRefGoogle Scholar
  19. Liu YH, Kao MC, Lai YL, Tsai JJ (2003) Efficacy of local nasal immunotherapy for Dp2-induced airway inflammation in mice: using Dp2 peptide and fungal immunomodulatory peptide. J Allergy Clin Immunol 112:301–310. doi:10.1067/mai.2003.1619 CrossRefGoogle Scholar
  20. Murasugi A, Tanaka S, Komiyama N, Iwata N, Kino K, Tsunoo H, Sakuma S (1991) Molecular cloning of a cDNA and a gene encoding an immunomodulatory protein, Ling Zhi-8, from a fungus, Ganoderma lucidum. J Biol Chem 266:2486–2493Google Scholar
  21. Paaventhan P, Jeremiah SJ, See VS, Shai V, Howard R, Kaw YC, Prasanna RK (2003) A 1.7 Å structure of Fve, a member of the new fungal immunomodulatory protein family. J Mol Biol 332:461–470. doi:10.1016/S0022-2836(03)00923-9 CrossRefGoogle Scholar
  22. Seow SV, Kuo IC, Paaventhan P, Kolatkar PR, Chua KY (2003) Crystallization and preliminary X-ray crystallographic studies on the fungal immunomodulatory protein Fve from the golden needle mushroom (Flammulina velutipes). Acta Crystallogr sect 59:1487–1489Google Scholar
  23. Strong DM, Ahmed AA, Thurman GB, Sell KW (1973) In vitro stimulation of murine spleen cells using a micro culture system and a multiple automated sample harvester. J Immunol Methods 2:279–291. doi:10.1016/0022-1759(73)90054-9 CrossRefGoogle Scholar
  24. Tanaka S, Ko K, Kino K, Tsuchiya K, Yamashita A, Murasugi S, Tsumoo H (1989) Complete amino acid sequence of an immunomodulatory protein, Ling Zhi-8. J Biol Chem 264:16372–16377Google Scholar
  25. Van der Hem LG, van der Vliet JA, Bocken CF, Kino K, Hoitsma AJ, Tax WJ (1995) Ling Zhi-8: studies of a new immunomodulating agent. Transplantation 60(5):438–443. doi:10.1097/00007890-199509000-00006 CrossRefGoogle Scholar
  26. Wang PH, Hsu CI, Tang YL, Lin JY, Ko JL (2004) Fungal immunomodulatory protein from Flammulina velutipes induces interferon-gamma production through p38 mitogen-activated protein kinase signaling pathway. J Agric Food Chem 52:2721–2725. doi:10.1021/jf034556s CrossRefGoogle Scholar
  27. Wermerskirchen AS, LaTocha DH, Clarle BL (2000) Adrenocorticotropic hormone controls concanavalin A activation of rat lymphocytes by modulating IL-2 production. J Life Sci 67:2177–2187. doi:10.1016/S0024-3205(00)00810-9 CrossRefGoogle Scholar
  28. Wu CM, Wu TY, Kao SS, Ko JL, Jinn TR (2008) Expression and purification of a recombinant Fip-fve from Flammulina velutipes in baculovirus-infected insect cells. J Appl Microbiol 104:1354–1362. doi:10.1111/j.1365-2672.2007.03686.x CrossRefGoogle Scholar
  29. Yeh CM, Yeh CK, Hsu XY, Luo QM, Lin MY (2008) Extracellular expression of a functional recombinant Ganoderma lucidum immunomodulatory protein by Bacillus subtilis and Lactococcus lactis. Appl Environ Microbiol 74:1039–1049. doi:10.1128/AEM.01547-07 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • Jing-Wei Lin
    • 1
  • Li-Xia Hao
    • 1
  • Gui-Xue Xu
    • 1
  • Fei Sun
    • 2
  • Feng Gao
    • 3
  • Ren Zhang
    • 4
  • Li-Xia Liu
    • 1
  1. 1.School of Life SciencesNortheast Normal UniversityChangchunChina
  2. 2.Institute of Frontier Medical Science of Jilin UniversityChangchunChina
  3. 3.Medical CenterDuke UniversityDurhamUSA
  4. 4.Department of Biological SciencesUniversity of WollongongWollongongAustralia

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