Jeyarajan Sivakumar, Peter Ansu Susan, Sathyan Aswathy, Ranjith Sukumar, Kandasamy Indira, Duraisamy Senbagam, Chidambaram Prahalathan, Kumarasamy Anbarasu
Microbial Biotechnology Laboratory, Department of Marine Biotechnology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India.
Department of Pharmacology, University of Michigan, Ann Arbor, MI, USA.
Appl Microbiol Biotechnol. 2024 Jan 26;108(1):176. doi: 10.1007/s00253-024-13017-5.
The demand for massive quantities of therapeutic active antimicrobial peptides (AMPs) is high due to their potential as alternatives to antibiotics. However, each antimicrobial peptide has unique properties, necessitating distinct synthesis and purification strategies for their large-scale production. In this study, we bio-synthesized and purified a functional enhanced variant of the AMP epinecidin-1, known as Ac-Var-1 (acid-cleavable variant-1). To generate the active peptide, we cloned the gene for Ac-Var-1 with acid-cleavable site (aspartic acid-proline) into the pET-32a expression vector, purified the fusion protein by His tag enrichment chromatography, and performed acid cleavage to release the active Ac-Var-1 peptide. After acid cleavage, the active Ac-Var-1 was purified and characterized by SDS-PAGE and mass spectrometry. The results from both techniques provided confirmation of the intactness of the purified Ac-Var-1. The Ac-Var-1 inhibited the growth of pathogenic Escherichia coli and Staphylococcus aureus. KEY POINTS : • Epinecidin-1 is a well-known antimicrobial peptide having multipotential bioactivities. • Epinecidin-1 variant is developed via the site-directed mutagenesis method to improve its structural stability and bioactivity. • AC-Var-1 development is an economical and easy method to remove peptide from tag protein.
由于抗菌活性肽（AMPs）有潜力成为抗生素的替代品，因此对大量治疗用活性抗菌肽的需求很高。然而，每种抗菌肽都有独特的性质，这就需要采用不同的合成和纯化策略来进行大规模生产。在本研究中，我们生物合成并纯化了一种功能性增强的抗菌肽表皮菌素-1变体，称为Ac-Var-1（酸可裂解变体-1）。为了产生活性肽，我们将带有酸可裂解位点（天冬氨酸-脯氨酸）的Ac-Var-1基因克隆到pET-32a表达载体中，通过His标签富集色谱法纯化融合蛋白，并进行酸裂解以释放活性Ac-Var-1肽。酸裂解后，对活性Ac-Var-1进行纯化，并通过SDS-PAGE和质谱进行表征。这两种技术的结果都证实了纯化后的Ac-Var-1的完整性。Ac-Var-1抑制了致病性大肠杆菌和金黄色葡萄球菌的生长。要点：•表皮菌素-1是一种具有多种生物活性的著名抗菌肽。•通过定点诱变方法开发表皮菌素-1变体，以提高其结构稳定性和生物活性。•AC-Var-1的开发是一种从标签蛋白中去除肽的经济且简便的方法。
