1. Molecular cloning, codon-optimized gene expression, and bioactivity assessment of two novel fungal immunomodulatory proteins from Ganoderma applanatum in Pichia
- Author
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Jingwei Lin, Xiao Han, Yan-Ye Ruan, Hui Ma, Xin Zhang, Shi-Xin Guan, Si-Ya Zhou, Wen-Li Fan, Zuo-wen Duan, Haoge Li, Li-Jing Chen, and Hangmei Liu
- Subjects
0106 biological sciences ,0301 basic medicine ,Agglutination ,Erythrocytes ,Cell Survival ,Ganoderma ,Molecular cloning ,01 natural sciences ,Applied Microbiology and Biotechnology ,Pichia ,law.invention ,Pichia pastoris ,Fungal Proteins ,Mice ,03 medical and health sciences ,Ganoderma applanatum ,law ,Gene Expression Regulation, Fungal ,010608 biotechnology ,Gene expression ,Animals ,Humans ,Immunologic Factors ,Cloning, Molecular ,Codon ,chemistry.chemical_classification ,biology ,General Medicine ,biology.organism_classification ,Recombinant Proteins ,Amino acid ,030104 developmental biology ,Biochemistry ,chemistry ,A549 Cells ,Recombinant DNA ,HeLa Cells ,Biotechnology - 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 IC50 of rFIP-gap1 towards A549 and HeLa cancer cells were 29.89 and 8.34 μg/mL, respectively, whereas IC50 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.
- Published
- 2018