Expression of Hybrid Peptide EF-1 in Pichia pastoris, Its Purification, and Antimicrobial Characterization.
Expression of Hybrid Peptide EF-1 in Pichia pastoris, Its Purification, and Antimicrobial Characterization.
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DOI：
10.3390/molecules25235538
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发表时间：
2020-11-26
期刊：
影响因子：
--
通讯作者：
Si D
中科院分区：
文献类型：
--
作者：
Li Z;Cheng Q;Guo H;Zhang R;Si D
EF-1是一种新的多肽，来源于两种细菌素，plantaricin E和plantaricin F。它对大肠杆菌有很强的抗菌活性，对红细胞的溶血作用可以忽略不计。然而，EF-1的化学合成受到其高成本的限制。在本研究中，我们建立了EF-1在毕赤酵母中的异源表达。转基因菌株成功表达了杂交EF-1肽，其分子量约为5 kDa。重组EF-1经Ni 2+亲和层析和反相高效液相色谱（RP-HPLC）纯化，得率为32.65 mg/L，纯度为94.9%。纯化的EF-1对革兰氏阳性菌和革兰氏阴性菌均表现出较强的抗菌和杀菌活性。碘化丙啶染色和扫描电镜观察表明，EF-1可直接诱导大肠杆菌细胞膜透化。杆菌因此，杂交EF-1不仅保留了亲本肽的个体特性，而且还获得了破坏革兰氏阴性菌膜的能力。同时，这种表达系统可以减少大规模肽生产的时间和成本，这确保了其在工业水平上的潜在应用。
EF-1 is a novel peptide derived from two bacteriocins, plantaricin E and plantaricin F. It has a strong antibacterial activity against Escherichia coli and with negligible hemolytic effect on red blood cells. However, the chemical synthesis of EF-1 is limited by its high cost. In this study, we established a heterologous expression of EF-1 in Pichia pastoris. The transgenic strain successfully expressed hybrid EF-1 peptide, which had a molecular weight of ~5 kDa as expected. The recombinant EF-1 was purified by Ni2+ affinity chromatography and reversed-phase high performance liquid chromatography (RP-HPLC), which achieved a yield of 32.65 mg/L with a purity of 94.9%. The purified EF-1 exhibited strong antimicrobial and bactericidal activities against both Gram-positive and -negative bacteria. Furthermore, propidium iodide staining and scanning electron microscopy revealed that EF-1 can directly induce cell membrane permeabilization of E. coli. Therefore, the hybrid EF-1 not only preserves the individual properties of the parent peptides, but also acquires the ability to disrupt Gram-negative bacterial membrane. Meanwhile, such an expression system can reduce both the time and cost for large-scale peptide production, which ensures its potential application at the industrial level.
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