Functional expression of FIP-fve, a fungal immunomodulatory protein from the edible mushroom Flammulina velutipes in Pichia pastoris GS115

• Published in: Journal of biotechnology Vol. 168; no. 4; pp. 527 - 533
• Main Authors: Lin, Jing-Wei, Jia, Jia, Shen, Yu-Hua, Zhong, Ming, Chen, Li-Jing, Li, Hao-Ge, Ma, Hui, Guo, Zhi-Fu, Qi, Ming-Fang, Liu, Li-Xia, Li, Tian-Lai
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.12.2013
• Subjects: ammonium sulfate; Animals; anticarcinogenic activity; Biological; biotechnology; cell viability; Chromatography; cultured cells; erythrocytes; Erythrocytes - drug effects; Flammulina - genetics; Flammulina - growth & development; Flammulina velutipes; Fungal immunomodulatory protein; Fungal Proteins - biosynthesis; Fungal Proteins - genetics; Fungal Proteins - pharmacology; Fungi; gel chromatography; Gene Expression Regulation - drug effects; Genes; Humans; immunomodulation; interferon-gamma; Interferon-gamma - secretion; interleukin-2; Interleukin-2 - secretion; lymphocytes; Lymphocytes - drug effects; Lymphocytes - secretion; methanol; Methyl alcohol; Mice; Mushrooms; Pichia - genetics; Pichia pastoris; polyacrylamide gel electrophoresis; Proteins; Recombinant proteins; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Recombinant Proteins - pharmacology; secretion; Secretions; Shake-flask; splenocytes; Western blotting; yeasts; Shake-flask; Flammulina velutipes; Recombinant proteins; Fungal immunomodulatory protein; Chromatography
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2013.09.013

FIP-fve is a bioactive protein isolated from the mushroom Flammulina velutipes, which belongs to the fungal immunomodulatory protein (FIP) family and demonstrates several kinds of biological activities including anti-allergy, anti-tumor and immunomodulation. In the current study, the FIP-fve gene was cloned and expressed in the yeast Pichia pastoris GS115, and its correctness was confirmed by SDS-PAGE and Western blot. Optimal expression of rFIP-fve was observed when the P. pastoris cells were cultured in 1% methanol for 96h, which resulted in a yield of 258.2mgl−1. The rFIP-fve protein was subsequently purified via ammonium sulfate precipitation and Sephadex G-100 gel chromatography. In vitro bioactivity examination showed that rFIP-fve could agglutinate human red blood cells and stimulate the cell viability of murine splenocytes. The immunomodulatory capacity and anti-tumor activity of rFIP-fve were demonstrated by enhanced interleukin-2 secretion and interferon-γ release from the murine lymphocytes, similar to the biological FIP-fve. In conclusion, the FIP-fve gene was functionally and effectively expressed in P. pastoris, and rFIP-fve displayed biological activities similar to those of native FIP-fve. These results indicated the potential use of rFIP-fve from P. pastoris as an effective and feasible source for therapeutic studies and medical applications.