Design, characterization and expression of a novel hybrid peptides melittin (1–13)-LL37 (17–30)
Design, characterization and expression of a novel hybrid peptides melittin (1–13)-LL37 (17–30)
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DOI：
10.1007/s11033-013-2900-0
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发表时间：
2014-05
影响因子：
2.8
通讯作者：
Rujuan Wu;Qing Wang;Zhaojun Zheng;Longmei Zhao;Yajing Shang;Xubiao Wei;Xiudong Liao;Rijun Zhang
中科院分区：
文献类型：
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作者：
Rujuan Wu;Qing Wang;Zhaojun Zheng;Longmei Zhao;Yajing Shang;Xubiao Wei;Xiudong Liao;Rijun Zhang
不同抗菌肽（AMPs）的杂交已成为获得具有更高抗菌活性但最小化细胞毒性的新型杂交AMPs的常见做法。结合melittin (M)的疏水n端片段和LL37 (L)的核心抗菌片段，首次设计了杂种肽melittin (1-13)-LL37 (17-30) (M -L)，分别对细菌和绵羊红细胞进行抑菌活性和溶血活性研究。结果表明，M - L对所有指示菌（尤其是革兰氏阳性菌）的抑菌活性均高于M和L，而对绵羊红细胞没有溶血活性，表明M - L可作为替代传统抗生素的潜在治疗药物。但是高昂的生物合成费用限制了其进一步的研究，因此M-L在大肠杆菌中进行了融合表达。大肠杆菌)过量生产杂交肽，从而解决问题。利用优选密码子将编码M-L的DNA序列克隆到pET-SUMO载体上，表达蛋白inE。coliBL21 (DE3)。IPTG诱导后，利用Ni-NTA Sepharose柱（纯度92%），每升发酵超声裂解液上清中回收约165 mg可溶性融合蛋白SUMO-M-L。经SUMO蛋白酶裂解和Ni-NTA Sepharose柱纯化后，每升培养得到23 mg重组M-L。综上所述，本研究不仅为杂交多肽M-L的过量生产提供了有效途径，而且为其进一步挖掘药物潜力和医学意义铺平了道路。
Hybridizing of different antimicrobial peptides (AMPs) has been a common practice for obtaining novel hybrid AMPs with elevated antibacterial activity but minimized cytotoxicity. The hybrid peptides melittin (1-13)-LL37 (17-30) (M–L) combining the hydrophobic N-teriminal fragment of melittin (M) with the core antibacterial fragment of LL37 (L), was designed for the first time to explore its antibacterial activity and hemolytic activity against bacteria and sheep erythrocyte respectively. Results showed that M–L had an even more potent antibacterial activity against all indicator strains (especially gram-positive bacteria) than M and L, whereas didn’t exhibit hemolytic activity to sheep erythrocytes, implying M–L can be served as a potential therapeutic drug to substitute traditional antibiotics. However the high expense of biosynthesis limited its further research, therefore fusion expression of M–L was carried out inEscherichia coli(E. coli) for overproducing the hybrid peptide so as to solve the problem. The DNA sequence encoding M–L with preferred codons was cloned into the pET-SUMO vector for protein expression inE. coliBL21 (DE3). After IPTG induction, approximately 165 mg soluble fusion protein SUMO-M–L was recovered per liter supernatant of the fermentation ultrasonic lysate using Ni–NTA Sepharose column (92 % purity). And 23 mg recombinant M–L was obtained per liter culture after cleavage of SUMO protease and purification of Ni–NTA Sepharose column. In sum, this research not only supplied an effective approach for overproducing hybrid peptide M–L, but paved the way for its further exploration on pharmaceutical potential and medical importance.