Functional expression of FIP-fve, a fungal immunomodulatory protein from the edible mushroom Flammulina velutipes in Pichia pastoris GS115.
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 9 6h, which resulted in a yield of 258.2 mg l(-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.
College of Horticulture, Shenyang Agriculture University, 120 Dongling Road, Shenyang, Liaoning Province 110866, China; College of Biological Science and Technology, Shenyang Agriculture University, 120 Dongling Road, Shenyang, Liaoning Province 110866, China.
Jing-Wei
JW
Lin
Jia
J
Jia
Yu-Hua
YH
Shen
Ming
M
Zhong
Li-Jing
LJ
Chen
Hao-Ge
HG
Li
Hui
H
Ma
Zhi-Fu
ZF
Guo
Ming-Fang
MF
Qi
Li-Xia
LX
Liu
Tian-Lai
TL
Li
D000818:Animals; D004912:Erythrocytes / Q000187:drug effects; D055393:Flammulina / Q000235:genetics* / Q000254:growth & development; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D007376:Interleukin-2 / Q000378:metabolism; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology
C547132:FIP-fve protein, Flammulina velutipes; D005656:Fungal Proteins; D007376:Interleukin-2; D011994:Recombinant Proteins; D007371:Interferon-gamma
Chromatography; Flammulina velutipes; Fungal immunomodulatory protein; Recombinant proteins; Shake-flask