Construction of a Pichia pastoris strain efficiently secreting irisin and assessment of its bioactivity in HepG2 cells
| Autor: | Jian-Hong Zhu, Huikun Duan, Muhammad Umar, Xingkai Yang, Minggang Li, Wenya Luo, Qiqi Liu, Xiaodan Li |
|---|---|
| Rok vydání: | 2018 |
| Předmět: |
Cell Survival
Apoptosis 02 engineering and technology Microscopy Atomic Force Biochemistry Pichia law.invention Pichia pastoris 03 medical and health sciences Structural Biology law Myokine Humans Viability assay Molecular Biology PI3K/AKT/mTOR pathway 030304 developmental biology Glucose Transporter Type 2 0303 health sciences biology Chemistry Biological activity General Medicine Hep G2 Cells 021001 nanoscience & nanotechnology biology.organism_classification Recombinant Proteins Cell biology Fibronectins Gene Expression Regulation Recombinant DNA biology.protein Hepatic stellate cell GLUT2 0210 nano-technology |
| Zdroj: | International journal of biological macromolecules. 124 |
| ISSN: | 1879-0003 |
| Popis: | Irisin, a circulating myokine, has been shown to effectively ameliorate insulin resistance and type 2 diabetes mellitus by administration of the recombinant protein. Therefore, it is important to efficiently produce active irisin protein and further characterize its potential mechanism against hepatic insulin resistance. In this study, we obtained a multi-copy irisin-expressing P. pastoris strain through an optimized method, which is pH 5.5, 1.8% methanol for 96 h, for producing a high amount of recombinant irisin protein following a series of screening and optimization procedures. The higher-glycosylated irisin, which is supposed to be the active form was obtained by dialysis and ion-exchange chromatography purification method. Both of the laser scanning confocal microscope and the atomic force microscope not only detected the high-effectiveness entering cells of FITC-irisin but also localized it on the membrane of HepG2 cells. Immunofluorescence staining further suggested that irisin could localize in the cytoplasm but not in the nucleus. We further showed that glycosylated irisin rescued palmitate-induced reduction in Glut2 expression and cell viability, inhibited the apoptosis, potentially by activating PI3K/AKT pathway. In summary, we developed an efficient irisin-expressing P. pastoris strain and optimal expression condition, visualized its distribution, demonstrated biological activity and potential mechanisms in hepatic cells. |
| Databáze: | OpenAIRE |
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