Expression, Purification, and Characterization of a Novel Hybrid Peptide with Potent Antibacterial Activity.
Expression, Purification, and Characterization of a Novel Hybrid Peptide with Potent Antibacterial Activity.
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具有有效抗菌活性的新型杂合肽的表达、纯化和表征
DOI：
10.3390/molecules23061491
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发表时间：
2018-06-20
期刊：
影响因子：
--
通讯作者：
Zhang R
中科院分区：
文献类型：
--
作者：
Wei X;Wu R;Zhang L;Ahmad B;Si D;Zhang R
从天蚕素A（C）和LL-37（L）序列衍生的杂合肽天蚕素A（1-8）-LL-37（17-30）（C-L）显示出比单独的C和L显著增加的抗菌活性和最小化的溶血活性。为了获得高水平的C-L表达，将编码C-L的DNA序列和优选密码子克隆到pET-SUMO中，构建融合表达载体，并在大肠杆菌（Escherichia coli，E. coli）BL21（DE3）。经Ni-NTA Sepharose柱纯化后，融合蛋白的产量为89.14 mg/L，纯度达92%。用SUMO-蛋白酶从融合蛋白上切下杂合C-L，得到17.54mg/L的活性C-L。此外，纯化的C-L具有与合成的C-L相同的抗菌和溶血活性。稳定性分析结果表明，在低于80 °C的温度下20 min，C-L的活性变化不大，但当温度超过80 °C时，C-L的活性显著下降。pH在5.0 ~ 10.0范围内变化对C-L的抗菌活性没有影响，但pH低于4.0时，C-L的抗菌活性迅速下降。在多种蛋白酶（胃蛋白酶、胰蛋白酶和蛋白酶K）的挑战下，C-L的功能活性保持在50%以上。本研究不仅为高水平生产杂合肽C-L提供了一条有效途径，而且为进一步探索杂合肽C-L在家畜乃至人类传染病防治中的应用奠定了基础。
The hybrid peptide cecropin A (1–8)–LL37 (17–30) (C–L), derived from the sequence of cecropin A (C) and LL-37 (L), showed significantly increased antibacterial activity and minimized hemolytic activity than C and L alone. To obtain high-level production of C–L, the deoxyribonucleic acid sequence encoding C–L with preferred codons was cloned into pET-SUMO to construct a fusion expression vector, and overexpressed in Escherichia coli (E. coli) BL21 (DE3). The maximum fusion protein (92% purity) was obtained with the yield of 89.14 mg/L fermentation culture after purification with Ni-NTA Sepharose column. The hybrid C–L was cleaved from the fusion protein by SUMO-protease, and 17.54 mg/L pure active C–L was obtained. Furthermore, the purified C–L showed identical antibacterial and hemolytic activity to synthesized C–L. Stability analysis results exhibited that the activity of C–L changed little below 80 °C for 20 min, but when the temperature exceeded 80 °C, a significant decrease was observed. Varying the pH from 5.0 to 10.0 did not appear to influence the activity of C–L, however, pH below 4.0 decreased the antibacterial activity of C–L rapidly. Under the challenge of several proteases (pepsin, trypsin, and proteinase K), the functional activity of C–L was maintained over 50%. In summary, this study not only supplied an effective approach for high-level production of hybrid peptide C–L, but paved the way for its further exploration in controlling infectious diseases of farm animals or even humans.
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DOI：
10.3390/molecules22122071
发表时间：
2017-12-13
期刊：
Molecules (Basel, Switzerland)
影响因子：
--
作者：
Pantic JM;Jovanovic IP;Radosavljevic GD;Arsenijevic NN;Conlon JM;Lukic ML
通讯作者：
Lukic ML
影响因子：
4.8
作者：
Papo, N;Shai, Y
通讯作者：
Shai, Y
影响因子：
3.6
作者：
LOW, AG
通讯作者：
LOW, AG
影响因子：
4.9
作者：
Saugar, JM;Rodríguez-Hernández, MJ;Rivas, L
通讯作者：
Rivas, L
影响因子：
1.6
作者：
Tomala, Magda;Lavrentieva, Antonina;Scheper, Thomas
通讯作者：
Scheper, Thomas