Expression and Purification of Hybrid LL-37Tα1 Peptide in Pichia pastoris and Evaluation of Its Immunomodulatory and Anti-inflammatory Activities by LPS Neutralization
This study pertains to the new approach for the development of hybrid peptide LL-37Tα1 and its biomedical applications. A linear cationic hybrid peptide, LL-37Tα1 was derived from two parental peptides (LL-37 and Tα1) recognized as potent anti-endotoxin without any hemolytic or cytotoxic activity. W...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2019-06-01
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| Series: | Frontiers in Immunology |
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| Online Access: | https://www.frontiersin.org/article/10.3389/fimmu.2019.01365/full |
| _version_ | 1819029796664901632 |
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| author | Baseer Ahmad Quratulain Hanif Quratulain Hanif Wei Xubiao Zhang Lulu Muhammad Shahid Muhammad Shahid Si Dayong Zhang Rijun |
| author_facet | Baseer Ahmad Quratulain Hanif Quratulain Hanif Wei Xubiao Zhang Lulu Muhammad Shahid Muhammad Shahid Si Dayong Zhang Rijun |
| author_sort | Baseer Ahmad |
| collection | DOAJ |
| description | This study pertains to the new approach for the development of hybrid peptide LL-37Tα1 and its biomedical applications. A linear cationic hybrid peptide, LL-37Tα1 was derived from two parental peptides (LL-37 and Tα1) recognized as potent anti-endotoxin without any hemolytic or cytotoxic activity. We successfully cloned the gene of hybrid peptide LL-37Tα1 in PpICZαA vector and expressed in the Pichia pastoris. The recombinant peptide was purified by Ni-affinity column and reverse-phase high performance liquid chromatography (RP-HPLC) with an estimated molecular mass of 3.9 kDa as determined by SDS-PAGE and mass spectrometry. We analyzed the LPS neutralization by limulus amebocyte lysate (LAL) activity and the results indicate that the hybrid peptide LL-37Tα1 directly binds endotoxin and significantly (p < 0.05) neutralizes the effect of LPS in a dose-dependent manner. Lactate dehydrogenase (LDH) assay revealed that LL-37Tα1 successfully reduces the LPS-induced cytotoxicity in mouse RAW264.7 macrophages. Moreover, it significantly (p < 0.05) decreased the levels of nitric oxide, proinflammatory cytokines including TNF-α, IL-6, IL-1β, and diminished the number of apoptotic cells in LPS-stimulated mouse RAW264.7 macrophages. Our results suggest that the P. pastoris expression system is cost-effective for commercial production of the immunomodulatory and anti-inflammatory hybrid peptide (IAHP) LL-37Tα1 and the peptide may serve as effective anti-endotoxin/anti-inflammatory agent with minimal cytotoxicity. |
| first_indexed | 2024-12-21T06:19:57Z |
| format | Article |
| id | doaj.art-3ed954c632a04ad08a8f53077c1b36c2 |
| institution | Directory Open Access Journal |
| issn | 1664-3224 |
| language | English |
| last_indexed | 2024-12-21T06:19:57Z |
| publishDate | 2019-06-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Immunology |
| spelling | doaj.art-3ed954c632a04ad08a8f53077c1b36c22022-12-21T19:13:17ZengFrontiers Media S.A.Frontiers in Immunology1664-32242019-06-011010.3389/fimmu.2019.01365458955Expression and Purification of Hybrid LL-37Tα1 Peptide in Pichia pastoris and Evaluation of Its Immunomodulatory and Anti-inflammatory Activities by LPS NeutralizationBaseer Ahmad0Quratulain Hanif1Quratulain Hanif2Wei Xubiao3Zhang Lulu4Muhammad Shahid5Muhammad Shahid6Si Dayong7Zhang Rijun8State Key Laboratory of Animal Nutrition and Feed Sciences, College of Animal Science and Technology, China Agricultural University, Beijing, ChinaState Key Laboratory of Animal Nutrition and Feed Sciences, College of Animal Science and Technology, China Agricultural University, Beijing, ChinaNational Institute for Biotechnology and Genetic Engineering, Pakistan Institute of Engineering and Applied Sciences, Faisalabad, PakistanState Key Laboratory of Animal Nutrition and Feed Sciences, College of Animal Science and Technology, China Agricultural University, Beijing, ChinaState Key Laboratory of Animal Nutrition and Feed Sciences, College of Animal Science and Technology, China Agricultural University, Beijing, ChinaState Key Laboratory of Animal Nutrition and Feed Sciences, College of Animal Science and Technology, China Agricultural University, Beijing, ChinaCollege of Veterinary Medicine, China Agricultural University, Beijing, ChinaState Key Laboratory of Animal Nutrition and Feed Sciences, College of Animal Science and Technology, China Agricultural University, Beijing, ChinaState Key Laboratory of Animal Nutrition and Feed Sciences, College of Animal Science and Technology, China Agricultural University, Beijing, ChinaThis study pertains to the new approach for the development of hybrid peptide LL-37Tα1 and its biomedical applications. A linear cationic hybrid peptide, LL-37Tα1 was derived from two parental peptides (LL-37 and Tα1) recognized as potent anti-endotoxin without any hemolytic or cytotoxic activity. We successfully cloned the gene of hybrid peptide LL-37Tα1 in PpICZαA vector and expressed in the Pichia pastoris. The recombinant peptide was purified by Ni-affinity column and reverse-phase high performance liquid chromatography (RP-HPLC) with an estimated molecular mass of 3.9 kDa as determined by SDS-PAGE and mass spectrometry. We analyzed the LPS neutralization by limulus amebocyte lysate (LAL) activity and the results indicate that the hybrid peptide LL-37Tα1 directly binds endotoxin and significantly (p < 0.05) neutralizes the effect of LPS in a dose-dependent manner. Lactate dehydrogenase (LDH) assay revealed that LL-37Tα1 successfully reduces the LPS-induced cytotoxicity in mouse RAW264.7 macrophages. Moreover, it significantly (p < 0.05) decreased the levels of nitric oxide, proinflammatory cytokines including TNF-α, IL-6, IL-1β, and diminished the number of apoptotic cells in LPS-stimulated mouse RAW264.7 macrophages. Our results suggest that the P. pastoris expression system is cost-effective for commercial production of the immunomodulatory and anti-inflammatory hybrid peptide (IAHP) LL-37Tα1 and the peptide may serve as effective anti-endotoxin/anti-inflammatory agent with minimal cytotoxicity.https://www.frontiersin.org/article/10.3389/fimmu.2019.01365/fullhybrid peptidesyeast expressionLPS neutralizationimmunomodulatoryapoptosis |
| spellingShingle | Baseer Ahmad Quratulain Hanif Quratulain Hanif Wei Xubiao Zhang Lulu Muhammad Shahid Muhammad Shahid Si Dayong Zhang Rijun Expression and Purification of Hybrid LL-37Tα1 Peptide in Pichia pastoris and Evaluation of Its Immunomodulatory and Anti-inflammatory Activities by LPS Neutralization Frontiers in Immunology hybrid peptides yeast expression LPS neutralization immunomodulatory apoptosis |
| title | Expression and Purification of Hybrid LL-37Tα1 Peptide in Pichia pastoris and Evaluation of Its Immunomodulatory and Anti-inflammatory Activities by LPS Neutralization |
| title_full | Expression and Purification of Hybrid LL-37Tα1 Peptide in Pichia pastoris and Evaluation of Its Immunomodulatory and Anti-inflammatory Activities by LPS Neutralization |
| title_fullStr | Expression and Purification of Hybrid LL-37Tα1 Peptide in Pichia pastoris and Evaluation of Its Immunomodulatory and Anti-inflammatory Activities by LPS Neutralization |
| title_full_unstemmed | Expression and Purification of Hybrid LL-37Tα1 Peptide in Pichia pastoris and Evaluation of Its Immunomodulatory and Anti-inflammatory Activities by LPS Neutralization |
| title_short | Expression and Purification of Hybrid LL-37Tα1 Peptide in Pichia pastoris and Evaluation of Its Immunomodulatory and Anti-inflammatory Activities by LPS Neutralization |
| title_sort | expression and purification of hybrid ll 37tα1 peptide in pichia pastoris and evaluation of its immunomodulatory and anti inflammatory activities by lps neutralization |
| topic | hybrid peptides yeast expression LPS neutralization immunomodulatory apoptosis |
| url | https://www.frontiersin.org/article/10.3389/fimmu.2019.01365/full |
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