High-level biosynthesis and purification of the antimicrobial peptide Kiadin based on non-chromatographic purification and acid cleavage methods
Abstract Antimicrobial peptides (AMPs) are renowned for their potent bacteriostatic activity and safety, rendering them invaluable in animal husbandry, food safety, and medicine. Despite their potential, the physiological toxicity of AMPs to host cells significantly hampers their biosynthetic produc...
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BMC
2025-01-01
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| Series: | Biotechnology for Biofuels and Bioproducts |
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| Online Access: | https://doi.org/10.1186/s13068-025-02607-8 |
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| author | Liangjun Zheng Fengyi Yang Chen Wang Muhammad Zafir Zishuo Gao Pilong Liu Fatma A. El-Gohary Xin Zhao Huping Xue |
| author_facet | Liangjun Zheng Fengyi Yang Chen Wang Muhammad Zafir Zishuo Gao Pilong Liu Fatma A. El-Gohary Xin Zhao Huping Xue |
| author_sort | Liangjun Zheng |
| collection | DOAJ |
| description | Abstract Antimicrobial peptides (AMPs) are renowned for their potent bacteriostatic activity and safety, rendering them invaluable in animal husbandry, food safety, and medicine. Despite their potential, the physiological toxicity of AMPs to host cells significantly hampers their biosynthetic production. This study presents a novel approach for the biosynthesis of the antimicrobial peptide Kiadin by engineering a DAMP4–DPS–Kiadin fusion protein to mitigate host cell toxicity and achieve high-level expression. Leveraging the unique properties of the DAMP4 protein, we developed a non-chromatographic purification method to isolate the DAMP4–DPS–Kiadin fusion protein with high purity. The instability of the D–P peptide bond under acidic conditions, combined with the thermal and saline stability of DAMP4, enabled efficient separation of Kiadin through acid cleavage and isoelectric precipitation, yielding Kiadin with 96% purity and a production yield of 29.3 mg/L. Our optimization of acid cleavage temperature, duration, and isoelectric precipitation conditions proved critical for maximizing the purification efficiency and expression levels of Kiadin. The biosynthesized Kiadin exhibited robust bacteriostatic activity against Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Bacillus cereus and Staphylococcus aureus. Notably, Kiadin demonstrated significant post-antibiotic effects by disrupting bacterial membrane integrity, inducing cytoplasmic leakage, and inhibiting biofilm formation in E. coli K88 and S. aureus Mu50, without cytotoxicity towards mouse macrophages. In vivo studies further confirmed Kiadin's exceptional therapeutic efficacy against abdominal infections caused by E. coli K88. The acid cleavage and non-chromatographic purification techniques developed in this study offer a cost-effective and efficient strategy for the high-purity production of AMPs. |
| format | Article |
| id | doaj-art-3b56d3ef9c9a42388f0e3967ca82444d |
| institution | Kabale University |
| issn | 2731-3654 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | BMC |
| record_format | Article |
| series | Biotechnology for Biofuels and Bioproducts |
| spelling | doaj-art-3b56d3ef9c9a42388f0e3967ca82444d2025-01-19T12:13:48ZengBMCBiotechnology for Biofuels and Bioproducts2731-36542025-01-0118111410.1186/s13068-025-02607-8High-level biosynthesis and purification of the antimicrobial peptide Kiadin based on non-chromatographic purification and acid cleavage methodsLiangjun Zheng0Fengyi Yang1Chen Wang2Muhammad Zafir3Zishuo Gao4Pilong Liu5Fatma A. El-Gohary6Xin Zhao7Huping Xue8Department of Animal Science and Technology, University of Northwest A&FDepartment of Animal Science and Technology, University of Northwest A&FDepartment of Animal Science and Technology, University of Northwest A&FDepartment of Animal Science and Technology, University of Northwest A&FDepartment of Animal Science and Technology, University of Northwest A&FDepartment of Animal Science and Technology, University of Northwest A&FDepartment of Hygiene and Zoonoses, Faculty of Veterinary Medicine, Mansoura UniversityDepartment of Animal Science, McGill UniversityDepartment of Animal Science and Technology, University of Northwest A&FAbstract Antimicrobial peptides (AMPs) are renowned for their potent bacteriostatic activity and safety, rendering them invaluable in animal husbandry, food safety, and medicine. Despite their potential, the physiological toxicity of AMPs to host cells significantly hampers their biosynthetic production. This study presents a novel approach for the biosynthesis of the antimicrobial peptide Kiadin by engineering a DAMP4–DPS–Kiadin fusion protein to mitigate host cell toxicity and achieve high-level expression. Leveraging the unique properties of the DAMP4 protein, we developed a non-chromatographic purification method to isolate the DAMP4–DPS–Kiadin fusion protein with high purity. The instability of the D–P peptide bond under acidic conditions, combined with the thermal and saline stability of DAMP4, enabled efficient separation of Kiadin through acid cleavage and isoelectric precipitation, yielding Kiadin with 96% purity and a production yield of 29.3 mg/L. Our optimization of acid cleavage temperature, duration, and isoelectric precipitation conditions proved critical for maximizing the purification efficiency and expression levels of Kiadin. The biosynthesized Kiadin exhibited robust bacteriostatic activity against Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Bacillus cereus and Staphylococcus aureus. Notably, Kiadin demonstrated significant post-antibiotic effects by disrupting bacterial membrane integrity, inducing cytoplasmic leakage, and inhibiting biofilm formation in E. coli K88 and S. aureus Mu50, without cytotoxicity towards mouse macrophages. In vivo studies further confirmed Kiadin's exceptional therapeutic efficacy against abdominal infections caused by E. coli K88. The acid cleavage and non-chromatographic purification techniques developed in this study offer a cost-effective and efficient strategy for the high-purity production of AMPs.https://doi.org/10.1186/s13068-025-02607-8Antimicrobial peptideBiosynthesisDAMP4Acid cleavageNon-chromatographic purification |
| spellingShingle | Liangjun Zheng Fengyi Yang Chen Wang Muhammad Zafir Zishuo Gao Pilong Liu Fatma A. El-Gohary Xin Zhao Huping Xue High-level biosynthesis and purification of the antimicrobial peptide Kiadin based on non-chromatographic purification and acid cleavage methods Biotechnology for Biofuels and Bioproducts Antimicrobial peptide Biosynthesis DAMP4 Acid cleavage Non-chromatographic purification |
| title | High-level biosynthesis and purification of the antimicrobial peptide Kiadin based on non-chromatographic purification and acid cleavage methods |
| title_full | High-level biosynthesis and purification of the antimicrobial peptide Kiadin based on non-chromatographic purification and acid cleavage methods |
| title_fullStr | High-level biosynthesis and purification of the antimicrobial peptide Kiadin based on non-chromatographic purification and acid cleavage methods |
| title_full_unstemmed | High-level biosynthesis and purification of the antimicrobial peptide Kiadin based on non-chromatographic purification and acid cleavage methods |
| title_short | High-level biosynthesis and purification of the antimicrobial peptide Kiadin based on non-chromatographic purification and acid cleavage methods |
| title_sort | high level biosynthesis and purification of the antimicrobial peptide kiadin based on non chromatographic purification and acid cleavage methods |
| topic | Antimicrobial peptide Biosynthesis DAMP4 Acid cleavage Non-chromatographic purification |
| url | https://doi.org/10.1186/s13068-025-02607-8 |
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