Gene cloning of a novel fungal immunomodulatory protein fromChroogomphis rutilusand its expression inPichia pastoris

Abstract

BACKGROUND Fungal immunomodulatory proteins (FIPs) are a newly identified protein family that exhibits immunomodulation and antitumor activities. However, little is known about the FIPs from Gomphidiaceae species. In this study, a novel FIP gene termed FIP‐cru was cloned from Chroogomphis rutilus, analyzed, and functionally expressed in Pichia pastoris GS115. In addtion, the bioactivity of rFIP‐cru was examined including hemagglutination, cytotoxicity, and interleukin‐2 assays in vitro. RESULTS FIP‐cru comprised 342 bp and encoded a peptide of 113 amino acids, which shared a high homology to other FIPs. rFIP‐cru was obtained by expressing the recombinant gene in P. pastoris GS115, with a yield of 148.5 mg L−1, and then purified via ammonium sulfate precipitation and agarose nickel magnetic nanobeads. The bioactivity assay showed that rFIP‐cru can agglutinate mouse and sheep red blood cells but not human cells. Additionally, rFIP‐cru can stimulate the proliferation of murine splenocytes and enhance the secretion of interleukin‐2. CONCLUSION FIP‐cru is a novel fungal immunomodulatory protein. The use of P. pastoris as an expression system will provide a feasible production method to obtain large amounts of rFIP‐cru to study its potential application in therapeutic and medical studies. © 2016 Society of Chemical Industry

Fungal immunomodulatory proteins in the context of biomedicine

“…From that date, six other FIPs in species of the genus Ganoderma have been reported: FIP-gts of G. tsugae (15), FIPgsi of G. sinensis (16), FIP -gja of G. japonicum (Genbank: AAX9824), FIP-gmi G. Microsporum (17), FIP-gas of G. atrum (GenBank: KM077027.1) and FIP-gap G. applanatum (Genbank: AEP68179). Other eleven fungal immunomodulatory proteins have been described at molecular or sequence level in other Basidiomycetes: FIP-fve of Flammulina velutipes (18), FIP-vvo of straw mushroom (19), FIP-Apl of Auricularia polytricha (20), FIP-aca of Antrodia camphorate (21), FIP-PCIP Pleurotus citrinopileatus (22), FIP-PCP of Poria cocos (23), FIP-tvc of Trametes versicolor (24,25), FIP-ppl Postia placenta (26), FIPcru Chroogomphis rutilus (27), FIP-Lrh of Lignosus rhinocerotis (Tiger milk mushroom) (28), FIP-tfu of Tremella fuciformis (GenBank: EF152774) and FIPnha Nectria haemotococca edge Ascomycetes (29).…”

“…Recently, a novel FIP gene from Chroogomphis rutilus (FIP-cru) was cloned, analyzed, and effectively expressed in P. pastoris GS115 (27). The recombinant rFIP-cru exhibited bioactivities, including the agglutination of mouse and sheep blood cells, the induction of murine splenocyte proliferation, and the enhancement of IL-2 secretion.…”

Enhanced heterologous expression of Trichoderma reesei Cel5A/Cel6A in Pichia pastoris with extracellular co‐expression of Vitreoscilla hemoglobin

“…Akbarzadeh et al . successfully expressed the codon optimized Trichoderma reesei Cel5A in Pichia pastoris and constructed the recombinant host capable of producing a maximum CMCase activity of 2 358 U mL ‐1 with 1% methanol induction for 72 h. Researchers have argued that the Pichia pastoris host appears a desirable candidate for the expression . Just recently, we also attempted the expression of codon optimized Trichoderma reesei Cel5A / Cel6A in Pichia pastoris GS115 under the AOX1 promoter .…”

“…Akbarzadeh et al 12 successfully expressed the codon optimized Trichoderma reesei Cel5A in Pichia pastoris and constructed the recombinant host capable of producing a maximum CMCase activity of 2 358 U mL -1 with 1% methanol induction for 72 h. Researchers have argued that the Pichia pastoris host appears a desirable candidate for the expression. 13,14 Just recently, we also attempted the expression of codon optimized Trichoderma reesei Cel5A / Cel6A in Pichia pastoris GS115 under the AOX1 promoter. 15,16 With a high-cell-density fed-batch (HCDFB) fermentation, two recombinants P. pastoris GS115-Cel5A/Cel6A presented high biomass and secretion level, producing 270 U mL -1 of Cel5A with 650 of OD 600 and 1100 U L -1 of Cel6A CMCase with 550 of OD 600 , respectively, suggesting that HCDFB cultivation is a favorable way to express the cellulase at high levels in the methanol inducible yeast.…”

Identification and Characterisation of a Novel Protein FIP-sch3 from Stachybotrys chartarum

“…Homologous cloning involves designing primers using known FIPs as templates and cloning FIPs in the same genus, so it is limited to identifying highly homologous proteins (identity>90%), making it difficult to further characterize FIP proteins. Until now, only five FIPs have been identified using the homologous cloning method: FIP-gmi [14], FIP-gsi from Ganoderma sinense [15], FIP-cru from Chroogomphis rutilus [16], FIP-gat from Ganoderma atrum [7], and LZ-9 [17]. Therefore, researchers have shifted away from the extraction or homologous cloning methods for identifying new FIPs and instead search for sequence similarity in the burgeoning genome databases.…”