Jeyarajan Sivakumar, Peter Ansu Susan, Ranjith Sukumar, Sathyan Aswathy, Duraisamy Senbagam, Kandasamy Indira, 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, Michigan, USA.
Biotechnol Appl Biochem. 2024 Dec;71(6):1384-1404. doi: 10.1002/bab.2637. Epub 2024 Jul 21.
Epinecidin-1 (epi-1), an antimicrobial peptide first identified in marine grouper fish, has multifunctional bioactivities. The present study aims to improve its therapeutic potential via structural modifications that could enhance its antimicrobial activity and stability. To achieve it, we replaced glycine and the first histidine in the parent epi-1 with lysine, which resulted in a peptide with a repeating KXXK motif and improved physiochemical properties related to antimicrobial activity. This modified peptide, referred to as glycine-to-lysine replaced-epi-1, also gained stability and a twofold increase in helical propensity. To produce the active peptide, overlap extension PCR was employed to generate the gene of GK-epi-1 via site-directed mutagenesis, which was then cloned into the pET-32a vector and expressed as a recombinant fusion protein in Escherichia coli C43 (DE3) strain. The recombinant protein was purified and digested with enterokinase to release the active peptide fragment, which was then evaluated for antimicrobial activity and stability. The lysine substitution led to an enhancement in broad-spectrum antimicrobial activity against a wide range of nosocomial pathogenic bacteria.
表没食子儿茶素-1（epi-1）是一种最初在海水石斑鱼中发现的抗菌肽，具有多种生物活性。本研究旨在通过结构修饰提高其治疗潜力，这些修饰可增强其抗菌活性和稳定性。为实现这一目标，我们将母体epi-1中的甘氨酸和第一个组氨酸替换为赖氨酸，得到了一种具有重复KXXK基序的肽，并改善了与抗菌活性相关的理化性质。这种修饰后的肽，称为甘氨酸到赖氨酸替换的epi-1，也获得了稳定性，螺旋倾向增加了两倍。为了生产活性肽，采用重叠延伸PCR通过定点诱变产生GK-epi-1基因，然后将其克隆到pET-32a载体中，并在大肠杆菌C43（DE3）菌株中表达为重组融合蛋白。重组蛋白经纯化后用肠激酶消化以释放活性肽片段，然后对其抗菌活性和稳定性进行评估。赖氨酸取代导致对多种医院病原菌的广谱抗菌活性增强。
