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

• Published in: BMC biotechnology 2018-12, Vol.18 (1), p.80-80, Article 80
• Main Authors: Qu, Zheng-Wei, Zhou, Si-Ya, Guan, Shi-Xin, Gao, Rui, Duan, Zuo-Wen, Zhang, Xin, Sun, Wei-Yan, Fan, Wen-Li, Chen, Shui-Sen, Chen, Li-Jing, Lin, Jing-Wei, Ruan, Yan-Ye
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; Amino acid sequence; Amino acids; Analogies; Animals; Biological activity; Cancer; Carcinoma; Cell Line; Chemical properties; Cloning; Conserved sequence; Cytokines; Cytokines - genetics; Cytokines - immunology; Cytotoxicity; Erythrocytes; Erythrocytes - drug effects; Erythrocytes - physiology; Feasibility studies; Fungal immunomodulatory protein; Fungal Proteins - genetics; Fungal Proteins - isolation & purification; Fungal Proteins - metabolism; Fungal Proteins - pharmacology; Fungi; Ganoderma; Ganoderma - chemistry; Ganoderma - genetics; Ganoderma - metabolism; Gene Expression; Genes; Genetic aspects; Genetic engineering; Genetic research; Hemagglutination Tests; Homology; Humans; Immunologic Factors - genetics; Immunologic Factors - isolation & purification; Immunologic Factors - metabolism; Immunologic Factors - pharmacology; Immunomodulation; Mice; Mushrooms; Phytochemicals; Pichia - genetics; Pichia - metabolism; Plant proteins; Proteins; Recombinant expression; Recombinant proteins; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Recombinant Proteins - pharmacology; Reishi; Sheep; Splenocytes; Stem cells; Sterols; Studies; Yeast
• ISSN: 1472-6750; 1472-6750
• DOI: 10.1186/s12896-018-0488-0

More than a dozen of fungal immunomodulatory proteins (FIPs) have been identified to date, most of which are from Ganoderma species. However, little is known about the similarities and differences between different Ganoderma FIPs' bioactivities. In the current study, two FIP genes termed FIP-gap1 and FIP-gap2 from G. applanatum, along with LZ-8 and FIP-gsi, another two representative Ganoderma FIP genes from G. lucidum and G. sinense were functionally expressed in Pichia. Subsequently, bioactivities of four recombinant Ganoderma FIPs were demonstrated and compared. All the four Ganoderma FIP genes could be effectively expressed in P. pastoris GS115 at expression levels ranging from 197.5 to 264.3 mg L and simply purified by one step chromatography using HisTrap™ FF prepack columns. Amino acid sequence analysis showed that they all possessed the FIP conserved fragments. The homologies of different Ganoderma FIPs were from 72.6 to 86.4%. In vitro haemagglutination exhibited that FIP-gap1, FIP-gsi and LZ-8 could agglutinate human, sheep and mouse red blood cells but FIP-gap2 agglutinated none. Besides, the immunomodulation activities of these Ganoderma FIPs were as: rFIP-gap2 > rFIP-gap1 > rLZ-8 and rFIP-gsi in terms of proliferation stimulation and cytokine induction on murine splenocytes. Additionally, the cytotoxic activity of different FIPs was: rFIP-gap1 > rLZ-8 > rFIP-gsi > rFIP-gap2, examined by their inhibition of three human carcinomas A549, Hela and MCF-7. Taken together, four typical Ganoderma FIP genes could be functionally expressed in P. pastoris, which might supply as feasible efficient resources for further study and application. Both similarities and differences were indeed observed between Ganoderma FIPs in their amino acid sequences and bioactivities. Comprehensively, rFIP-gaps from G. applanatum proved to be more effective in immunomodulation and cytotoxic assays in vitro than rLZ-8 (G. lucidum) and rFIP-gsi (G. sinense).