Fusion of a Novel Native Signal Peptide Enhanced the Secretion and Solubility of Bioactive Human Interferon Gamma Glycoproteins in Nicotiana benthamiana Using the Bamboo Mosaic Virus-Based Expression System
Plant viruses may serve as expression vectors for the efficient production of pharmaceutical proteins in plants. However, the downstream processing and post-translational modifications of the target proteins remain the major challenges. We have previously developed an expression system derived from...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2020-11-01
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| Series: | Frontiers in Plant Science |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fpls.2020.594758/full |
| _version_ | 1818116087227613184 |
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| author | Min-Chao Jiang Chung-Chi Hu Chung-Chi Hu Wei-Li Hsu Tsui-Ling Hsu Na-Sheng Lin Yau-Heiu Hsu Yau-Heiu Hsu |
| author_facet | Min-Chao Jiang Chung-Chi Hu Chung-Chi Hu Wei-Li Hsu Tsui-Ling Hsu Na-Sheng Lin Yau-Heiu Hsu Yau-Heiu Hsu |
| author_sort | Min-Chao Jiang |
| collection | DOAJ |
| description | Plant viruses may serve as expression vectors for the efficient production of pharmaceutical proteins in plants. However, the downstream processing and post-translational modifications of the target proteins remain the major challenges. We have previously developed an expression system derived from Bamboo mosaic virus (BaMV), designated pKB19, and demonstrated its applicability for the production of human mature interferon gamma (mIFNγ) in Nicotiana benthamiana. In this study, we aimed to enhance the yields of soluble and secreted mIFNγ through the incorporation of various plant-derived signal peptides. Furthermore, we analyzed the glycosylation patterns and the biological activity of the mIFNγ expressed by the improved pKB19 expression system in N. benthamiana. The results revealed that the fusion of a native N. benthamiana extensin secretory signal (SSExt) to the N-terminal of mIFNγ (designated SSExt mIFNγ) led to the highest accumulation level of protein in intracellular (IC) or apoplast washing fluid (AWF) fractions of N. benthamiana leaf tissues. The addition of 10 units of ‘Ser-Pro’ motifs of hydroxyproline-O-glycosylated peptides (HypGPs) at the C-terminal end of SSExt mIFNγ (designated SSExt mIFNγ(SP)10) increased the solubility to nearly 2.7- and 1.5-fold higher than those of mIFNγ and SSExt mIFNγ, respectively. The purified soluble SSExt mIFNγ(SP)10 protein was glycosylated with abundant complex-type N-glycan attached to residues N56 and N128, and exhibited biological activity against Sindbis virus and Influenza virus replication in human cell culture systems. In addition, suspension cell cultures were established from transgenic N. benthamiana, which produced secreted SSExt mIFNγ(SP)10 protein feasible for downstream processing. These results demonstrate the applicability of the BaMV-based vector systems as a useful alternative for the production of therapeutic proteins, through the incorporation of appropriate fusion tags. |
| first_indexed | 2024-12-11T04:16:56Z |
| format | Article |
| id | doaj.art-04ee91ab17904b758aa6c13b5b9884e6 |
| institution | Directory Open Access Journal |
| issn | 1664-462X |
| language | English |
| last_indexed | 2024-12-11T04:16:56Z |
| publishDate | 2020-11-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Plant Science |
| spelling | doaj.art-04ee91ab17904b758aa6c13b5b9884e62022-12-22T01:21:13ZengFrontiers Media S.A.Frontiers in Plant Science1664-462X2020-11-011110.3389/fpls.2020.594758594758Fusion of a Novel Native Signal Peptide Enhanced the Secretion and Solubility of Bioactive Human Interferon Gamma Glycoproteins in Nicotiana benthamiana Using the Bamboo Mosaic Virus-Based Expression SystemMin-Chao Jiang0Chung-Chi Hu1Chung-Chi Hu2Wei-Li Hsu3Tsui-Ling Hsu4Na-Sheng Lin5Yau-Heiu Hsu6Yau-Heiu Hsu7Ph.D. Program in Microbial Genomics, National Chung Hsing University and Academia Sinica, Taichung, TaiwanGraduate Institute of Biotechnology, National Chung Hsing University, Taichung, TaiwanAdvanced Plant Biotechnology Center, National Chung Hsing University, Taichung, TaiwanGraduate Institute of Microbiology and Public Health, College of Veterinary Medicine, National Chung Hsing University, Taichung, TaiwanGenomics Research Center, Academia Sinica, Taipei, TaiwanInstitute of Plant and Microbial Biology, Academia Sinica, Taipei, TaiwanGraduate Institute of Biotechnology, National Chung Hsing University, Taichung, TaiwanAdvanced Plant Biotechnology Center, National Chung Hsing University, Taichung, TaiwanPlant viruses may serve as expression vectors for the efficient production of pharmaceutical proteins in plants. However, the downstream processing and post-translational modifications of the target proteins remain the major challenges. We have previously developed an expression system derived from Bamboo mosaic virus (BaMV), designated pKB19, and demonstrated its applicability for the production of human mature interferon gamma (mIFNγ) in Nicotiana benthamiana. In this study, we aimed to enhance the yields of soluble and secreted mIFNγ through the incorporation of various plant-derived signal peptides. Furthermore, we analyzed the glycosylation patterns and the biological activity of the mIFNγ expressed by the improved pKB19 expression system in N. benthamiana. The results revealed that the fusion of a native N. benthamiana extensin secretory signal (SSExt) to the N-terminal of mIFNγ (designated SSExt mIFNγ) led to the highest accumulation level of protein in intracellular (IC) or apoplast washing fluid (AWF) fractions of N. benthamiana leaf tissues. The addition of 10 units of ‘Ser-Pro’ motifs of hydroxyproline-O-glycosylated peptides (HypGPs) at the C-terminal end of SSExt mIFNγ (designated SSExt mIFNγ(SP)10) increased the solubility to nearly 2.7- and 1.5-fold higher than those of mIFNγ and SSExt mIFNγ, respectively. The purified soluble SSExt mIFNγ(SP)10 protein was glycosylated with abundant complex-type N-glycan attached to residues N56 and N128, and exhibited biological activity against Sindbis virus and Influenza virus replication in human cell culture systems. In addition, suspension cell cultures were established from transgenic N. benthamiana, which produced secreted SSExt mIFNγ(SP)10 protein feasible for downstream processing. These results demonstrate the applicability of the BaMV-based vector systems as a useful alternative for the production of therapeutic proteins, through the incorporation of appropriate fusion tags.https://www.frontiersin.org/articles/10.3389/fpls.2020.594758/fullinterferon gammasignal peptidesanti-virus activityplant viral vectorglycosylationBamboo mosaic virus |
| spellingShingle | Min-Chao Jiang Chung-Chi Hu Chung-Chi Hu Wei-Li Hsu Tsui-Ling Hsu Na-Sheng Lin Yau-Heiu Hsu Yau-Heiu Hsu Fusion of a Novel Native Signal Peptide Enhanced the Secretion and Solubility of Bioactive Human Interferon Gamma Glycoproteins in Nicotiana benthamiana Using the Bamboo Mosaic Virus-Based Expression System Frontiers in Plant Science interferon gamma signal peptides anti-virus activity plant viral vector glycosylation Bamboo mosaic virus |
| title | Fusion of a Novel Native Signal Peptide Enhanced the Secretion and Solubility of Bioactive Human Interferon Gamma Glycoproteins in Nicotiana benthamiana Using the Bamboo Mosaic Virus-Based Expression System |
| title_full | Fusion of a Novel Native Signal Peptide Enhanced the Secretion and Solubility of Bioactive Human Interferon Gamma Glycoproteins in Nicotiana benthamiana Using the Bamboo Mosaic Virus-Based Expression System |
| title_fullStr | Fusion of a Novel Native Signal Peptide Enhanced the Secretion and Solubility of Bioactive Human Interferon Gamma Glycoproteins in Nicotiana benthamiana Using the Bamboo Mosaic Virus-Based Expression System |
| title_full_unstemmed | Fusion of a Novel Native Signal Peptide Enhanced the Secretion and Solubility of Bioactive Human Interferon Gamma Glycoproteins in Nicotiana benthamiana Using the Bamboo Mosaic Virus-Based Expression System |
| title_short | Fusion of a Novel Native Signal Peptide Enhanced the Secretion and Solubility of Bioactive Human Interferon Gamma Glycoproteins in Nicotiana benthamiana Using the Bamboo Mosaic Virus-Based Expression System |
| title_sort | fusion of a novel native signal peptide enhanced the secretion and solubility of bioactive human interferon gamma glycoproteins in nicotiana benthamiana using the bamboo mosaic virus based expression system |
| topic | interferon gamma signal peptides anti-virus activity plant viral vector glycosylation Bamboo mosaic virus |
| url | https://www.frontiersin.org/articles/10.3389/fpls.2020.594758/full |
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