Molecular cloning, codon-optimized gene expression, and bioactivity assessment of two novel fungal immunomodulatory proteins from Ganoderma applanatum in Pichia

Abstract

Fungal immunomodulatory proteins (FIPs) have been identified from a series of fungi, especially in Ganoderma species. However, little is known about the FIPs from G. applanatum. In this study, two novel FIP genes, termed as FIP-gap1 and FIP-gap2, were cloned from G. applanatum, characterized and functionally expressed after codon optimization in Pichia pastoris GS115. Results showed that FIP-gap1 and FIP-gap2 comprised 342-bp encoding peptides of 113 amino acids, which shared a high homology with other Ganoderma FIPs. The yield of recombinant FIP-gap1 and FIP-gap2 increased significantly after codon optimization and reached 247.4 and 197.5 mg/L, respectively. Bioactivity assay in vitro revealed that both rFIP-gap1 and rFIP-gap2 could agglutinate mouse, sheep, and human red blood cells. Besides, rFIP-gap1 and rFIP-gap2 obviously stimulated the proliferation of mouse splenocytes and enhanced IL-2 and IFN-γ release. Cytotoxicity detection indicated that IC of rFIP-gap1 towards A549 and HeLa cancer cells were 29.89 and 8.34 μg/mL, respectively, whereas IC of rFIP-gap2 to the same cancer cells were 60.92 and 41.05 μg/mL, respectively. Taken together, novel FIP gaps were cloned and functionally expressed in P. pastoris, which can serve as feasible and stable resources of rFIP gaps for further studies and potential applications.

Recombinant Expression and Bioactivity Comparison of Four Typical Fungal Immunomodulatory Proteins from Three Main Ganoderma Species

“…And the IC 50 value of another FIP, FIP-sch3 ( Stachybotrys chartarum ), was 10.80 μg/mL [29]. Besides, the cytotoxicity of rFIP-gaps towards A549 and Hela cells in current study was accordant with those in previous reports with very similar IC 50 values [35].…”

“…Ever since the first FIP, known as LZ-8 or FIP-glu, was isolated from the mycelia of G. lucidum by Kino et al in last century [24], more than fifteen FIPs have been identified from different fungi to date [25–31]. Nine of those FIPs are from Ganoderma species including LZ-8 (FIP-glu, G. lucidum ), LZ-9 ( G. lucidum ), FIP-gts ( G. tsugae ), FIP-gja ( G. japonicum ), FIP-gsi ( G. sinense ), FIP-gmi ( G. microsporum ), FIP-gas ( G. astum ), FIP-gbo ( G. boninense ), FIP-gat ( G. atrum ), FIP-gap1 and FIP-gap2 (both from G. applanatum ) [32–35]. The FIPs consist of 110–114 amino acids with molecular weights of 12–13 kDa, which are rich in Asp and Val but poor in His, Cys and Met.…”

“…Ganoderma . Similarly, a pair of novel FIP genes have also cloned from G. applanatum , the typical species of subgenus Elfringia , in our previous study [34, 35], which were termed as FIP-gap1 (or known as FIP-gap , Genbank No. JN167598.1) and FIP-gap2 ( Genbank No.…”

Identification and Functional Characterization of a Novel Immunomodulatory Protein From Morchella conica SH

“…FIP-glu can enhance the transcription of IL-2, IL-3, IL-4, IFN-g, and TNF-a (60). FIP-gts (Ganoderma tsugae) can induce cytokine secretion, and FIP-gaps enhances IL-2 and IFN-g release from mouse lymphocytes (23). However, FIP-gmi from Ganoderma microsporum exhibits the effect of down regulation of TNF-a (28).…”

Characterization of a novel fungal immunomodulatory protein, FIP-SJ75 shuffled from Ganoderma lucidum , Flammulina velutipes and Volvariella volvacea

“…The first FIP, designated as FIP-glu (LZ-8), was isolated from Ganoderma lucidum (Kino et al, 1989). Until now, FIPs have been isolated and identified from several fungi, such as Flammulina velutipes (FIP-fve) (Ko, Hsu, Lin, Kao, & Lin, 1995), Volvariella volvacea (FIP-vvo) (Hsu, Hsu CI, Kao, & Lin, 1997), Auricularia polytricha (FIP-app) (Sheu, Chien, Chien, Chen, & Chin, 2004), Ganoderma microsporum (FIP-gmi) (Wu et al, 2007), Ganoderma sinense (FIP-gsi) (Zhou, Xie, Hong, & Li, 2009), Poria cocos (FIP-pcp) (Chang, Yeh, & Sheu, 2009), Trametes versicolor (FIP-tvc) (Li, Wen, Liu, Zhou, & Chen, 2012), Nectria haematococca (FIP-nha) (Bastiaan-Net et al, 2013), Ganoderma lucidum (LZ-9) (Bastiaan-Net et al, 2013), Postia placenta (FIP-ppl) (Li, Shi, Ding, Nie, & Tang, 2015), Ganoderma atrum (FIP-gat) (Xu et al, 2016), Chroogomphis rutilus (FIP-cru) (Lin et al, 2016), Dichomitus squalens (FIP-dsq2) (Li et al, 2017), Ganoderma applanatum (FIP-gap) (Zhou et al, 2018), and Lentinus tigrinus (FIP-lti) (Gao et al, 2019). A protein family called FIPs was composed of these proteins (Li et al, 2011).…”